Monoclonal antibodies reacting with pellicular antigens of Toxoplasma gondii tachyzoites have been selected among hybridomas produced against this organism by immunofluorescence assay. These antigens have been further characterized by immunofluorescence on living zoites, Western immunoblotting and immunoprecipitation of lactoperoxidase surface radio-iodinated tachyzoite lysates. The simultaneous characterization of 5 different surface antigens (P43, P35, P30, P23, P22) some of which have already been studied individually allowed a better definition of these antigens and the characterization of a yet undescribed surface molecule (P23).

Toxoplasma gondii is known to induce specific behavioural changes in its intermediate hosts. This is usually considered to be an evolutionary adaptation aimed to increase the probability of transmission of the parasite into its definitive host, the cat, by predation. In rodents an increase of reaction time as well as many other specific behavioural patterns have been observed. Here we report the results of our double blind study showing the significantly longer reaction times of 60 subjects with latent toxoplasmosis in comparison with those of 56 controls. Moreover, the existence of a positive correlation between length of infection and mean reaction time suggested that slow and cumulative effects of latent toxoplasmosis rather than a one-step (and possibly transient) effect of acute toxoplasmosis disease are responsible for the decrease of psychomotor performance of infected subjects. To our knowledge, this is the first study confirming the existence of such parasite-induced changes in human behaviour that could be considered in evolutionary history of the human species as adaptive from the point of view of parasite transmission.

The skin site at which ticks attach to their hosts to feed is the critical interface between the tick and its host, and tick-borne pathogens. This site is highly modified by the pharmacologically active molecules secreted in tick saliva. For pathogens, it is an ecologically privileged niche that many exploit. Such exploitation is referred to as saliva-activated transmission (SAT) – the indirect promotion of tick-borne pathogen transmission via the actions of bioactive tick saliva molecules on the vertebrate host. Here we review evidence for SAT and consider what are the most likely candidates for SAT factors among the tick pharmacopoeia of anti-haemostatic, anti-inflammatory and immunomodulatory molecules identified to date. SAT factors appear to differ for different pathogens and tick vector species, and possibly even depend on the vertebrate host species. Most likely we are searching for a suite of molecules that act together to overcome the redundancy in host response mechanisms. Whatever they turn out to be, the quest to identify the tick molecules that mediate SAT is an exciting one, and offers new insights to controlling ticks and tick-borne diseases.

Leishmania are protozoan parasites spread by a sandfly insect vector and causing a spectrum of diseases collectively known as leishmaniasis. The disease is a significant health problem in many parts of the world resulting in an estimated 12 million new cases each year. Current treatment is based on chemotherapy, which is difficult to administer, expensive and becoming ineffective due to the emergence of drug resistance. Leishmaniasis is considered one of a few parasitic diseases likely to be controllable by vaccination. The relatively uncomplicated leishmanial life cycle and the fact that recovery from infection renders the host resistant to subsequent infection indicate that a successful vaccine is feasible. Extensive evidence from studies in animal models indicates that solid protection can be achieved by immunisation with protein or DNA vaccines. However, to date no such vaccine is available despite substantial efforts by many laboratories. Advances in our understanding of Leishmania pathogenesis and generation of host protective immunity, together with the completed Leishmania genome sequence open new avenues for vaccine research. The major remaining challenges are the translation of data from animal models to human disease and the transition from the laboratory to the field. This review focuses on advances in anti-leishmania vaccine development over the recent years and examines current problems hampering vaccine development and implementation.

Human fascioliasis infection sources are analysed for the first time in front of the new worldwide scenario of this disease. These infection sources include foods, water and combinations of both. Ingestion of freshwater wild plants is the main source, with watercress and secondarily other vegetables involved. The problem of vegetables sold in uncontrolled urban markets is discussed. Distinction between infection sources by freshwater cultivated plants, terrestrial wild plants, and terrestrial cultivated plants is made. The risks by traditional local dishes made from sylvatic plants and raw liver ingestion are considered. Drinking of contaminated water, beverages and juices, ingestion of dishes and soups and washing of vegetables, fruits, tubercles and kitchen utensils with contaminated water are increasingly involved. Three methods to assess infection sources are noted: detection of metacercariae attached to plants or floating in freshwater, anamnesis in individual patients, and questionnaire surveys in endemic areas. The infectivity of metacercariae is reviewed both under field conditions and experimentally under the effects of physicochemical agents. Individual and general preventive measures appear to be more complicated than those considered in the past. The high diversity of infection sources and their heterogeneity in different countries underlie the large epidemiological heterogeneity of human fascioliasis throughout.

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterizing the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd-stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70[ratio ]30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effect of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW <2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 μg/ml, each inhibited significantly the migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restored larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin.

A number of published studies of competition between parasite species are examined and compared. It is suggested that two general levels of interaction are discernible: these correspond to the two levels of competition recognized by workers studying free-living animals and plants: ‘exploitation’ and ‘interference’ competition. The former may be defined as the joint utilization of a host species by two or more parasite species, while the latter occurs when antagonistic mechanisms are utilized by one species either to reduce the survival or fecundity of a second species or to displace it from a preferred site of attachment. Data illustrating both levels of interaction are collated from a survey of the published literature and these suggest that interference competition invariably operates asymmetrically. The data are also used to estimate a number of population parameters which are important in determining the impact of competition at the population level. Theoretical models of host-parasite associations for both classes of competition are used to examine the expected patterns of population dynamics that will be exhibited by simple two-species communities of parasites that utilize the same host population. The analysis suggests that the most important factor allowing competing species of parasites to coexist is the statistical distribution of the parasites within the host population. A joint stable equilibrium should be possible if both species are aggregated in their distribution. The size of the parasite burdens at equilibrium is then determined by other life-history parameters such as pathogenicity, rates of resource utilization and antagonistic ability. Comparison of these theoretical expectations with a variety of sets of empirical data forms the basis for a discussion about the importance of competition in natural parasite populations. The models are used to assess quantitatively the potential for using competing parasite species as biological control agents for pathogens of economic or medical importance. The most important criterion for identifying a successful control agent is an ability to infect a high proportion of the host population. If such a parasite species also exhibits an intermediate level of pathology or an efficient ability to utilize shared common resources, antagonistic interactions between the parasite species contribute only secondarily to the success of the control. Competition in parasites is compared with competition in free-living animals and plants. The comparison suggests further experimental tests which may help to assess the importance of competition in determining the structure of more complex parasite-host communities.

Semi-defined and defined media for the growth of culture forms of Trypanosoma brucei sspp. have been developed by enrichment of tissue culture medium 199 with additional vitamins, amino acids, salts and other compounds. The semi-defined medium contains an acid hydrolysate of casein: in the empirically devised defined medium the casein requirement has been circumvented by inclusion of additional vitamins and amino acids. Both media are very hypertonic. Control of pH was found to be particularly critical for growth. The optimum temperature for growth in the semidefined medium was between 25°C and 28°C, but cells would undergo one or two division cycles at 37°C.

The nature of parasitism at the population level is defined in terms of the parasite's influence on the natural intrinsic growth rate of its host population. It is suggested that the influence on this rate is related to the average parasite burden/host and hence to the statistical distribution of parasites within the host population.

Theoretical models of host–parasite associations are used to assess the regulatory influence of parasitic species on host population growth. Model predictions suggest that three specific groups of population processes are of particular importance: over-dispersion of parasite numbers/host, density dependence in parasite mortality or reproduction and parasite-induced host mortality that increases faster than linearly with the parasite burden. Other population mechanisms are shown to have a destabilizing influence, namely: parasite-induced reduction in host reproductive potential, direct parasite reproduction within the host and time delays in the development of transmission stages of the parasite.

These regulatory and destabilizing processes are shown to be commonly observed features of natural host-parasite associations. It is argued that interactions in the real world are characterized by a degree of tension between these regulatory and destabilizing forces and that population rate parameter values in parasite life-cycles are very far from being a haphazard selection of all numerically possible values. It is suggested that evolutionary pressures in observed associations will tend to counteract a strong destabilizing force by an equally strong regulatory influence. Empirical evidence is shown to support this suggestion in, for example, associations between larval digeneans and molluscan hosts (parasite-induced reduction in host reproductive potential counteracted by tight density-dependent constraints on parasite population growth), and interactions between protozoan parasites and mammalian hosts (direct parasite reproduction counteracted by a well-developed immunological response by the host).

The type of laboratory and field data required to improve our understanding of the dynamical properties of host–parasite population associations is discussed and it is suggested that quantitative measurement of rates of parasite-induced host mortality, degrees of over-dispersion, transmission rates and reproductive and mortality rates of both host and parasite would provide an important first step. The value of laboratory work in this area is demonstrated by reference to studies which highlight the regulatory influence of parasitic species on host population growth.

Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2·5–25 µl of splenic aspirate or of 50–500 µl of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2·5 × 10−7 µl of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.

The longevity and infectivity of isolated populations of Plasmodium falciparum gametocytes were studied. Following chloroquine treatment gametocyte numbers fell with a constant rate of loss over a period of 16–24days; the populations had a half-life of 2·4 days. The sex ratio stayed constant throughout at 4 female:1 male. The ability of the microgametocytes to exflagellate and the infectivity of the population to mosquitoes persisted for 3 weeks. Antibodies to the gametocytes were detected but not in every patient studied. It was concluded that the gametocytes of P. falciparum are both long-lived and show persistent infectivity to mosquitoes. They can stimulatae antibody production but the immune response appears to play no part in their elimination, which probably takes place in the spleen as a part of the normal process of removing old, damaged and malformed red cells.

The multilaminate vesicles present in the tegument cytoplasm appear to fuse with side channels projecting out into the cytoplasm from the base of the surface pits. Their lamellate contents then unroll and spread out to form a trilaminate membranocalyx lining the pits and covering the tegument surface. The plant lectin concanavalin A appears to stabilize the process of vesicle fusion leading to an aggregation of multilaminate vesicles trapped in the lumen of the surface pits. The membranocalyx can be labelled with cationized ferritin. Chase incubations in normal medium indicate that by 4 h most of the label and the membranocalyx to which it is bound have been lost to the medium. This suggests that under the conditions of these experiments the membranocalyx has a half-life of 2–3 h.

There is a pressing need to develop novel anti-schistosomal drugs, as current treatment relies largely on praziquantel (PZQ). To further strengthen current evidence of the anti-schistosomal properties of mefloquine (MQ), we studied the temporal effect of this compound in vitro and in vivo, and examined alterations on the tegumental surface of schistosomula and adults of S. mansoni by means of scanning electron microscopy (SEM). Schistosomula and adults were each incubated in vitro using MQ over a wide concentration range (1–100 μg/ml). In addition, mice infected with adult S. mansoni were treated with a single oral dose of 400 mg/kg MQ, and worms were recovered 24, 48, 72, 96 and 120 h following treatment. MQ showed a rapid onset of action on schistosomula in vitro; 100 and 75 μg/ml of MQ killed schistosomula immediately; the minimal lethal and effective concentrations of MQ on schistosomula after 1 h were 25 and 5 μg/ml, respectively. Adult worms incubated with 100 and 10 μg/ml of MQ were dead after 1 h and 24 h of incubation, respectively. A hepatic shift of adult schistosomes was observed in mice already 24 h after treatment, and 120 h following treatment >98% of all worms had translocated to the liver. SEM observations revealed extensive tegumental destruction, including blebbing, shrinking and sloughing, particularly following in vitro incubation and on the tegument of female worms.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deaths per year, along with the emergence of multi- and extensively-drug resistant strains of Mtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent ‘Achilles heel’ that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.

Birds harbouring several malarial parasites are common in the wild, and resolving such multiple infections is important for our understanding of host–parasite relationships. We propose a simple and reasonably accurate method for detecting and resolving multiple infections, based on the analysis of parasite cytochrome b DNA sequences: genetically mixed infections are first identified by double nucleotide peaks on sequence electropherograms, and later retrieved by TA-cloning. We applied this method to wild birds, and to experimentally created mixes with varying proportion of two parasites (Plasmodium spp. and Haemoproteus spp.). In general, the method was very efficient in detecting and resolving multiple infections, but some problems were encountered. Several multiple infections were erroneously scored as simple, either because one of the parasite lineages was a better target for the primers used, or because it was much more abundant in the mix. On the other hand, single nucleotide substitutions and template switching during PCR produced artificial sequences in some clones. We discuss the utility of the method, and propose a framework for its use when screening for genetically diverse avian malarial parasites.

Eggs of an immune suppressive strain ( = virulent) of the parasitoid Leptopilina boulardi are encapsulated neither in resistant nor in susceptible strains of Drosophila melanogaster but are encapsulated in Drosophila yakuba. Eggs of a non-immune suppressive strain ( = avirulent) of the same parasitoid are encapsulated in a resistant strain of D. melanogaster and in D. yakuba but are not encapsulated in a susceptible strain of D. melanogaster. Egg chorion in the 2 parasitoid strains showed the same morphology and the same modifications after egg laying whatever the host strain. When a capsule is built, a small dotted dense layer was first spread on the chorion, followed by accumulation layers of cells (plasmatocytes and lamellocytes) and lastly necrosis of the inner haemocytes. The encapsulated eggs darken only at the time of necrosis of haemocytes. In susceptible hosts, neither the tiny dense layer nor haemocyte accumulation occured. We concluded that (1) this tiny dense layer was present before the deposition of the first haemocytes, (2) inhibition of deposition of this dense layer was the initial event of the induced immunosupression, (3) haemocytes other than lamellocytes were engaged in caspsule formation, (4) the immunosupressive factors did not target only the lamellocytes but also the plasmatocytes, (5) darkening of the encapsulated eggs was due to cell necrosis rather than to extracellular melanin deposition.

Toxoplasma gondii, a cosmopolitan protozoan parasite, is known to induce behavioural alterations in rodents and may exert an effect on human personality and behaviour. The mechanism of parasite-induced alterations in host behaviour has not been described, but it was hypothesized that development of Toxoplasma tissue cysts in the brain could affect the dopaminergic neuromodulatory system. In this study, we tested the effect of latent Toxoplasma infection on mouse behaviour associated with activity of the dopaminergic system, i.e. locomotion in a novel environment and exploration test. Additionally, we examined the behavioural response of Toxoplasma-infected mice to a selective dopamine uptake inhibitor, GBR 12909. In both genders, Toxoplasma infection decreased locomotion in the open field. Infected females displayed an increased level of exploration in the holeboard test. GBR 12909 induced suppression in holeboard-exploration in the infected males, but had an opposite effect on the controls. These results suggest an association between Toxoplasma gondii infection and changes in the dopaminergic neuromodulatory system.

Accurate identification of aspermic Fasciola forms in Japan remains difficult because of their morphological variations. In order to characterize the forms genetically, nucleotide sequences of ribosomal internal transcribed spacer (ITS1 and ITS2) and mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes in 34 liver flukes from 16 prefectures in Japan were analysed. Two major forms represented by Fsp 1 and Fsp 2 had sequences identical to or closely resembling those of F. hepatica and F. gigantica, respectively, in all the 4 DNA markers and were mainly distributed in northern and eastern-western parts of Japan, respectively. Fsp 1 and Fsp 2 would have been introduced into Japan with infected cattle of 2 distinct lineages via the Korean Peninsula and spread through limited parts of Japan (northern and eastern-western parts) together with the movement of each cattle lineage. The Japanese form (Fsp 1/2), which showed heterozygosity in ribosomal DNA and Fsp 2 haplotype in mitochondrial DNA, may have originated in interspecific cross hybridization between paternal F. hepatica and maternal F. gigantica.

Before the 1990s, human fascioliasis diagnosis focused on individual patients in hospitals or health centres. Case reports were mainly from developed countries and usually concerned isolated human infection in animal endemic areas. From the mid-1990s onwards, due to the progressive description of human endemic areas and human infection reports in developing countries, but also new knowledge on clinical manifestations and pathology, new situations, hitherto neglected, entered in the global scenario. Human fascioliasis has proved to be pronouncedly more heterogeneous than previously thought, including different transmission patterns and epidemiological situations. Stool and blood techniques, the main tools for diagnosis in humans, have been improved for both patient and survey diagnosis. Present availabilities for human diagnosis are reviewed focusing on advantages and weaknesses, sample management, egg differentiation, qualitative and quantitative diagnosis, antibody and antigen detection, post-treatment monitoring and post-control surveillance. Main conclusions refer to the pronounced difficulties of diagnosing fascioliasis in humans given the different infection phases and parasite migration capacities, clinical heterogeneity, immunological complexity, different epidemiological situations and transmission patterns, the lack of a diagnostic technique covering all needs and situations, and the advisability for a combined use of different techniques, at least including a stool technique and a blood technique.

The Drugs for Neglected Diseases initiative (DNDi) has defined and implemented an early discovery strategy over the last few years, in fitting with its virtual R&D business model. This strategy relies on a medium- to high-throughput phenotypic assay platform to expedite the screening of compound libraries accessed through its collaborations with partners from the pharmaceutical industry. We review the pragmatic approaches used to select compound libraries for screening against kinetoplastids, taking into account screening capacity. The advantages, limitations and current achievements in identifying new quality series for further development into preclinical candidates are critically discussed, together with attractive new approaches currently under investigation.