An attempt was made to determine the relative importance of the different life-cycle stages of Eimeria maxima in the induction of immunity and also those stages most affected by the immune response of the host. In one experiment the life-cycle was controlled by chemotherapy but in all other experiments partial life-cycles were induced by transfers of infected mucosa between hosts.

The results indicated that the second generation schizont stage is probably that most concerned in the induction of protective immunity and that sexual stages are most susceptible to immune inhibition. After initial inhibition in the immune host the earlier asexual stages were able to resume development when transferred to a susceptible host. The longer the period of exposure to the immune environment, the less able was the parasite to recover.

The capacity of polymorphonuclear neutrophils (PMN) and other leucocytes of the innate immune system to expel their DNA in a controlled process into the extracellular environment to trap and kill pathogenic microorganisms led to a paradigm shift in our comprehension of host leucocyte-pathogen interactions. Formation of neutrophil extracellular traps (NETs) has recently been recognized as a novel effector mechanism of the host innate immune response against microbial infections. Meanwhile evidence has arisen that NET formation is a widely spread mechanism in vertebrates and invertebrates and extends not only to the entrapment of microbes, fungi and viruses but also to the capture of protozoan and metazoan parasites. PMN produce NETs after stimulation with mitogens, cytokines or pathogens in a controlled process which depends on reactive oxygen species (ROS) and the induction of the Raf-MEK-ERK-mediated signalling pathway cascade. NETs consist of nuclear DNA as a backbone decorated with histones, antimicrobial peptides, and PMN-specific granular enzymes thereby providing an extracellular matrix capable of entrapping and killing invasive pathogens. This review is intended to summarize parasite-related data on NETs. Special attention will be given to NET-associated mechanisms by which parasites, in particular apicomplexa, might be hampered in their ability to reproduce within the host cell and complete the life cycle.

To assess the importance of cattle as a source of human cryptosporidial infections in Slovenia, Cryptosporidium isolates from calves and humans with cryptosporidiosis were characterized genetically by direct DNA sequencing, targeting a variable region of the 60 kDa glycoprotein (gp60) gene. In total, 15 genetic variants, designated ‘subtypes’, were identified, of which 7 were novel. In humans, C. hominis Ia (subtype IaA17R3) and Ib (IbA10G2) and Cryptosporidium parvum IIa (IIaA9G1R1, IIaA11G2R1, IIaA13R1, IIaA14G1R1, IIaA15G1R1, IIaA15G2R1, IIaA16G1R1, IIaA17G1R1 and IIaA19G1R1), IIc (IIcA5G3), and IIl (IIlA16R2) were recorded; this is the first record of the latter subtype in humans. In cattle, C. parvum IIa (IIaA13R1, IIaA15G2R1, IIaA16R1 and IIaA16G1R1) and IIl (IIlA16R2 and IIlA18R2) were recorded. Of the 15 subtypes identified, subtypes of C. parvum IIa were the most frequently encountered (>90%) in both humans and calves. The present findings suggest that zoonotic transmission plays an important role in sporadic human cryptosporidiosis in Slovenia.

The results of the studies on our model combination Trichobilharzia ocellata–Lymnaea stagnalis, presented in this review, lead to the conclusion that schistosomes use multiple strategies to reach their goals, i.e. to propagate and to continue their life cycle. They have to escape from being attacked by the internal defence system (IDS) of the snail host and to profoundly affect the host's energy flow, of which reproduction and growth are the main determinants, for their own benefit. These physiological changes they establish mainly by interfering with the two regulatory systems in the snail host, the IDS and the neuroendocrine system (NES). Moreover, these two regulatory systems clearly interact with each other. Parasitic E/S products affect the host's IDS both in a direct and an indirect way. The neuropeptides or neuropeptide-like substances that are secreted by parasite glands into the host directly suppress haemocyte activity in the snail. The indirect effects include effects of (1) peptides from connective tissue cells and (2) neuropeptides from NES and/or IDS. Parasitic E/S products also induce the effects on energy flow in the host. These E/S products act either directly on a target, as shown for the inhibiting effect of the parasite on the development of the male copulation organ, or on the NES regulating reproductive activity, e.g. on gene expression. Indirect effects of E/S products on the NES (hormone-receptor interaction, electrical activity) are mediated by a factor from connective tissue cells, presumably belonging to the IDS. The physiological changes in the snail host are obviously of vital importance for the parasites, since they make use of different strategies to bring them about.

Tick toxicoses, of which paralysis is the most widespread and dominant form, are important elements of pathogenesis induced by ticks. Tick paralysis is the most widespread and dominant form of tick toxicoses. Non-paralytic forms of tick toxicoses do occur and evidence suggests that these forms of toxicoses are not evolutionary related. While functional significance has been suggested for tick toxins, the advantages for tick survival in general are not clear. This review considers the molecular nature of tick toxins, the possibility that tick toxins have originated more than once independently and whether these toxins could have unrecognized benign functions.

The bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode, closely related to O. volvulus, the causal agent of human River Blindness, and, sharing with it, the same vector. This brief review, based on a presentation at the BSP Autumn Symposium 1999, describes recent work supported by the WHO Drug Development Research Macrofil programme and the Edna McConnell Clark Foundation vaccine development programme, to research the chemotherapy and immunology of onchocerciasis utilising this model system, with experimental infections in Liverpool and field infections in northern Cameroon. In a series of chemotherapeutic trials involving 10 compounds in 20 treatment regimes, the comparability of drug efficacy against O. ochengi with that described against O. volvulus has been demonstrated. Repeated, long-term treatment with oxytetracycline has been shown to be macrofilaricidal and the effect is hypothesized to be related to action on Wolbachia endobacteria, abundant in O. ochengi. Avermectins/milbemycins are not macrofilaricidal (even in high and repeated long-term treatments) but induce sustained abrogation of embryogenesis. In prospective, field exposure experiments with naive calves, prophylactic treatments with ivermectin and moxidectin prevented the development of adult worm infection, raising the possibility that drug-attenuated larval challenge infections may induce immunity. Putatively immune adult cattle exist in endemically exposed populations, and these have been shown to be significantly less susceptible to challenge than age-matched naive controls, whereas radically drug-cured, previously patently-infected cattle were not. Experimental infections with O. ochengi have revealed the kinetics of the immune response in relation to parasite development and demonstrate analogous responses to those reported in O. volvulus infection in humans and chimpanzees. In an immunization experiment with irradiated L3 larvae, cattle were significantly protected against experimental challenge – the first such demonstration of the experimental induction of immunity in a natural Onchocerca host–parasite system. Taken collectively, these studies not only demonstrate the similarity between the host–parasite relationships of O. ochengi in cattle and O. volvulus in humans, but promise to advance options for the control of human onchocerciasis.

The archaeological evidence for parasites in the Roman era is presented in order to demonstrate the species present at that time, and highlight the health consequences for people living under Roman rule. Despite their large multi-seat public latrines with washing facilities, sewer systems, sanitation legislation, fountains and piped drinking water from aqueducts, we see the widespread presence of whipworm (Trichuris trichiura), roundworm (Ascaris lumbricoides) and Entamoeba histolytica that causes dysentery. This would suggest that the public sanitation measures were insufficient to protect the population from parasites spread by fecal contamination. Ectoparasites such as fleas, head lice, body lice, pubic lice and bed bugs were also present, and delousing combs have been found. The evidence fails to demonstrate that the Roman culture of regular bathing in the public baths reduced the prevalence of these parasites. Fish tapeworm was noted to be widely present, and was more common than in Bronze and Iron Age Europe. It is possible that the Roman enthusiasm for fermented, uncooked fish sauce (garum) may have facilitated the spread of this helminth. Roman medical practitioners such as Galen were aware of intestinal worms, explaining their existence and planning treatment using the humoural theory of the period.

An electrophysiological preparation of Ascaris suum pharyngeal muscle suitable for recording changes of input conductance using a 2-microelectrode current clamp and pharmacological study is described. The preparation is shown to contain a glutamate-gated Cl (ion sensitive) channel sensitive to the avermectin analogue, milbemycin D. The application of glutamate produces a dose-dependent increase in Cl conductance and the effect of glutamate is potentiated by milbemycin D. Milbemycin D also produced a dose-dependent increase in input conductance.

Proliferative kidney disease (PKD), caused by the malacosporean parasite Tetracapsuloides bryosalmonae, causes significant losses among salmonids in Western Europe and North America. The role of salmonid fish in the life-cycle of this parasite has been conjectured upon for over a quarter of a century. To examine whether fish can transmit the infection to bryozoans, the known invertebrate host, water containing parasitized brown trout Salmo trutta was pumped into tanks containing colonies of Fredericella sultana collected from the wild. The specific parasite-free status of these colonies being first assessed, by PCR and prolonged laboratory culture. After 6 weeks exposure to the brown trout aquarium effluent, portions of these colonies displayed overt infections with T. bryosalmonae. This was in contrast to control bryozoans, derived from the experimental colonies prior to exposure, which remained T. bryosalmonae negative. In addition, spores obtained from the experimentally infected colonies were exposed to naïve rainbow trout, resulting in clinical PKD, thus completing a cycle of transmission. During the experiments, the infection was noted to inhibit statoblast formation within bryozoans and appeared to be pathogenic, finally killing the bryozoan host. These findings indicate that fish can transmit the parasite to bryozoans and are an integral part of this parasite's life-cycle.

Analysis of the electrical properties of neurons in the motornervous system of Ascaris suum suggests that it is largely an analogue system. The motorneurons do not conduct action potentials and they release transmitter tonically at their normal resting potential; transmitter release is increased or decreased as a continuous function of membrane potential. Despite extensive physiological descriptions of the electrical properties of the neurons and their synapses, as well as morphological descriptions of the synaptic circuitry of the system, the predicted activities of the neurons in the circuit differ from those observed by direct recording in semi-intact behaving animals. We conclude that the description of the circuit is incomplete. There are several possibilities for the missing elements, including chemical, proprioceptive, and additional neuronal components. Recently, attention has been focussed most heavily on the intercellular chemical signalling systems; in addition to those mediated by classical neurotransmitters, a surprisingly complex array of neuropeptides has been identified. One family of these peptides, the AF peptides, has been analyzed in detail. It comprises at least 20 peptides, and they fall into sequence-related subfamilies. One of these subfamilies, containing 6 peptides, is encoded by a single transcript, suggesting that the AF peptides are under multiple genetic control. All AF peptides tested have potent activity on the motornervous system and or on muscle. There are multiple physiological activities, and cellular localization studies show multiple patterns of cellular expression. Studies on Panagrellus and Caenorhabditis emphasize the diversity of this family and its genetic control.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.

We present a simulation population model for the African tick Rhipicephalus appendiculatus, based on previous analyses of the mortality factors most closely correlated with observed population changes at 11 sites in equatorial and South Africa. The model incorporates temperature-dependent rates of egg production and development, climate-driven density-independent mortality rates, particularly during the adult-larval stage, and density-dependent regulation of both nymphs and adults. Diapause is also included for tick populations in southern Africa. The model successfully describes both the seasonality and annual range of variation in numbers of each tick stage observed at each of 4 test sites in Uganda, Burundi and South Africa. Sensitivity analysis showed that the final version of the model is robust to 4-fold variation in most parameter values (that were per force based on informed guesses), but is more sensitive to the regression coefficients determining density-dependent interstadial mortality (that were derived from analysis of field data). The model is able to predict the seasonality of ticks from a site in Kenya where a full prior population analysis was not possible because only adults and nymphs had been counted. The model is potentially applicable to other species of ticks, both tropical and temperate, to predict tick abundance and seasonality as risk factors for tick-borne diseases.

Excystment of the metacercaria of Fasciola hepatica is an active process and occurs in two stages—activation and emergence. Activation is initiated by high concentrations of carbon dioxide, reducing conditions and a temperature about 39 °C. The reducing conditions increase the rate of action of the other two stimuli. The carbon dioxide stimulus need only be applied for 5 min, but the exposure time to the reducing conditions has to be of the order of 30 min. Changes in the order of application of the stimuli carbon dioxide and redox potential have no effect.

The second phase, emergence, is triggered by bile. Metacercariae were held in an activated condition for 24 h, and when bile was added emergence took place normally.

During excystment the metacercariae exhibit a complex behaviour pattern. After activation there is an initial period of rotatory activity, but after about 20 min a quiescent phase ensues when the metacercariae contract away from the cyst wall at an imperceptible rate. This behaviour occurs in response to stimulus from a high concentration of carbon dioxide and a temperature about 39 °C. When the metacercariae are exposed to bile the second phase of activity is initiated, consisting of antero-posterior thrusting movements directed against the ventral side of the cyst wall. Within about 15 min the juvenile flukes escape through a small circular hole in the ventral surface of the cyst wall which corresponds to the ventral plug region.

A technique for the hatching and subsequent in vitro cultivation of the oncospheres of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis to cystic larvae is described. The cytology and histology of developing larvae were also studied.

The basic culture medium consisted of medium 858 containing additional glucose and K+ supplemented with the appropriate host sera. Fresh sera from young animals was generally more successful in promoting growth than sera from commercial sources. Cultivation was carried out at 37°C in roller tubes with a gas phase of 10% O2+5% CO2 in N2.

The most successful results were obtained with T. pisiformis, oncospheres of which were grown to the cystic stage with development of hooks and suckers. During the later culture period, the bladder, although morphologically normal, ‘collapsed’ and did not retain its cystic shape.

In culture, larvae of T. pisiformis became motile at 3 days and at 5 days began secreting droplets from a glandular apical region; this region stained differentially in alcian blue.

Some larvae of T. pisiformis showed a tendency to undergo transverse or longitudinal fission, an effect possibly associated with the centrifugation applied during the early cultivation procedures.

Early cystic development was obtained with the other species studied, but experiments carried out were only of a preliminary nature. However, the development rate in these experiments approximated that recorded in vivo.

It is now becoming generally recognized that there are three different amoebae which may be present in the human bowel—the harmless Entamoeba coli(Lösch) Schaudinn and “Entamoeba” nana Wenyon and O'Connor, and the pathogenic E. histolytica Schaudinn. We have discussed these three species in an earlier paper (1917), and there noted their chief distinctive characters. In the present paper we shall describe a fourth and much less common species which we have recently encountered. It differs in some respects so conspicuously from the others that it appears to us necessary to place it not only in a new species, but even in a new genus. We propose therefore the new name Dientamoeb fragilis for our amoeba, but we can discuss the question of nomenclature most conveniently after we have described the organism itself. This, therefore, we shall now do.

In a previous paper (MacLeod, 1934) the different physical factors in the environmental complex of the sheep tick were discussed in their relation to the development of the gorged tick, and an experimental analysis was made of the effects produced, under laboratory conditions, by their action and interaction. The present paper deals with the active unfed ticks. These present rather a different problem from the gorged ticks, for, whereas in their case the climatic optimum resolved itself into that combination of conditions under which development proceeded favourably and with a low attendant mortality, the unfed tick, on the other hand, is faced with two desiderata, survival and hostparasitisation, the physical optima for which may not be coincident. It is necessary, therefore, to consider not only the climatic optimum for survival and activity, but also the factor or factors which influence parasitisation, and those conditions under which host-parasitisation is facilitated.

Dans ce travail je me propose d'étudier la biologie et la morphologic des larves des Anthomyides, appartenant surtout au groupe éthologique de carnivores. Certaines de ces larves étaient déjà connues auparavant, mais les caractères essentiels de leur morphologic manquaient dans leur description; nos documents sur les autres se réduisaient à leur signalement dans un tel ou tel milieu et enfin quelques unes de ces larves étaient totalement inconnues.

Two sets of oligonucleotide primers, one derived from a repetitive sequence and the other from the gene encoding a 67 kDa sporozoite antigen of Theileria parva, were used to amplify parasite DNA from the blood of T. parva-infected carrier cattle using the polymerase chain reaction (PCR). PCR amplification products were obtained from 15 carrier cattle infected with one of 4 different T. parva stocks. Successful amplifications were performed using DNA from 2 cattle infected with T. p. parva Pemba Mnarani, 10 cattle infected with T. p. parva Marikebuni, 2 cattle infected with T. p. bovis Boleni and 1 animal infected with T. p. lawrencei 7014. No amplification products were obtained from any of 7 cattle which had been infected with the T. p. parva Muguga stock. A synthetic oligonucleotide, which hybridized specifically to T. p. parva Marikebuni DNA among 6 T. parva stocks tested, was designed using sequence data from within the region of the T. parva genome amplified by the repetitive sequence primers. The oligonucleotide was used to probe PCR products and to increase the sensitivity and specificity of carrier animal detection. Southern blot analysis using a T. parva repetitive sequence probe demonstrated the existence of restriction fragment length polymorphisms between parasites isolated from T. p. parva Marikebuni-infected carrier cattle. The use of the PCR and other methods of carrier animal detection are discussed.

The DNA of a parasite is the ultimate blueprint of that parasite, the one characteristic which normally remains unchanged during every stage of the life-cycle. All the DNA sequence in the egg of a species of parasite are also in the larvae and adults of the same species. The same DNA is present in the parasite whether it is in a free-living stage, in an invertebrate vector or in a vertebrate host such as man. The molecular basis for DNA diagnosis is to allow labelled single-stranded species or strain-specific DNA sequences, selected from well-characterized reference species, to find and hybridize with homologous DNA from, or in, the unknown isolates of parasites. DNA probes are now available for most vector borne parasitic diseases. Parasitological identification problems are mostly concerned with distinguishing closely related strains or subspecies, for example detecting Taenia solium eggs as opposed to T. saginata eggs, or finding which of the 15 man-infecting subspecies of Leishmania is present in a single cutaneous lesion, the commonest clinical sign of the disease, or in a sandfly. For efficient hybridization by the present methods there has to be enough of a particular sequence present in a parasite's genome to make a feasible target. Therefore, DNA probes for parasites have been selected from repetitive, reiterated or multicopy DNA with intrinsic extensive sequence variation. DNA, which is free of coding restraint, can evolve rapidly to give differences between species, so that introns, ribosome gene spacers, variant genes, pseudo-genes and non-conserved DNA have all been used for DNA diagnosis. The major problems of sequence selection have been greatly aided by the use of recombinant DNA methods, which have the added advantage of economical production of DNA probes. The unique characteristics of kinetoplast mini-circle DNA in Leishmania has allowed the selection of a complex species, subspecies, strain and even isolate-specific DNA probes. These have been used successfully for Southern filter endonuclease fragment DNA identification, for dot-blot recognition of less than 200 parasites and non-radioactive detection of DNA sequence homology by ‘in situ’ hybridization and light microscopy in a single Leishmania cell. The adaptation of the forensic human genetic fingerprinting technique has allowed identification of L. braziliensis DNA in human biopsy material, even the presence of a vast excess of human DNA. Fingerprinting with ribosomal spacer-derived recombinant DNA probes has been used to discriminate Echinococcus species. The identification of Taenia species has been accomplished using probes from a genomic library of size-selected DNA fragments, and synthetic oligonucleotides are available for Onchocerca subspecies detection. The new technologies combining repeated genomic sequence probes with pulse field separation of the chromosomes of parasites, has opened up new avenues of research. Double probes for the simultaneous identification of the insect vector and the carried parasite have recently been reported. Lastly, the polymerase chain reaction technique has given us the opportunity of amplifying even single copy genes in one parasite to give sufficient DNA for positive identification. DNA diagnosis in parasitology is now on a par with the DNA diagnosis of viruses and human genetic disorders. The last ten years have seen many exciting developments in DNA diagnosis; the next years should see DNA diagnosis routinely used in epidemiological and clinical situations in countries where parasitic diseases are a major public health problem.