A method for phenetic analysis of karyotype data has been developed for Leishmania populations. Measurement of size difference between chromosomes recognized by a given DNA probe in different isolates led to the formulation of a Chromosome Size Difference Index (CSDI). The method was applied to phenetic analysis of 4 sets of chromosomes – each set being recognized by a different probe – in 37 L. (Viannia) peruviana isolates sampled along a North–South transect through the Peruvian Andes and, in 11 L. (V.) braziliensis isolates from the Amazonian forest (Peru, Bolivia and Brazil). Karyotype variability was better accounted for by CSDI than by a method based on disjunctive encoding of karyotype data. CSDI evidenced the nature of relationships between L. braziliensis and L. peruviana and it provided a coherent picture of geographical and genomic differentiation among parasite populations. The latter did cluster according to their geographical origin. L. braziliensis was found karyotypically more homogeneous than L. peruviana. Within L. peruviana, Northern populations were closer to L. braziliensis than to Southern L. peruviana populations. The validity of karyotypic populations, or karyodemes, was sustained.

The causal prophylactic activity of the novel hydroxynaphthoquinone, S66C80, was assessed against the exo-erythrocytic (EE) stages of Plasmodium berghei cultured in the human hepatoma cell line, HepG2. 566C80 was found to be highly active as an inhibitor of EE development and was more active than the established causal prophylactic pyrimethamine. A 566C80 concentration of 1·85 × 10−9 M, added 3 h after sporozoite invasion, reduced the numbers of EE forms visible at 48 h by 50%, while the equivalent concentration of pyrimethamine was 1·95 × 10−8 M.

1. An examination of the cuticle of Oxyuris equi has revealed the presence of previously unidentified structures. The layers of the cuticle of this nematode have been renamed.

2. The cuticle of Strongylus equinus has been examined in a similar fashion and, whilst the basic nomenclature of the various layers has been retained, previous descriptions of the individual layers have been modified and extended.

3. Further evidence is put forward in favour of the presence of pore canals in the cuticle of Ascaris lumbricoides.

Clones of single oocysts, single sporocysts, single sporozoites, single proliferative parasites, single cysts and single cystozoites of Isospora (Toxoplasma) gondii (KB-strain) were made under visual control using a de Fonbrune micromanipulator. Cloning was successful in 28, 32, 21, 8, 54 and 7% of the trials, respectively. All clones were used for monoclonal infection in non-immune conventionally reared (CV) or specified pathogen-free (SPF) cats. Pre-patent and patent periods, sporulation percentages of excreted oocysts, mouse infectivity of sporulated oocysts, antibody response and immunity to reinfection of CV cats were determined. For these parameters almost no differences were observed between monoclonal infections and infections described with the non-cloned KB-strain. In all cats autopsied during the patent period, 5–8 days post-infection, macrogametes, microgametes and oocysts were found. Since meiosis occurs during sporulation and since all free-living and intermediate host stages proved to be bisexual, it is concluded that sex differentiation in I. (T) gondii is not determined by segregation of sex chromosomes or sex genes but is caused by some final host factor(s) that induce(s) differential gene expression in genetically identical cells.

In recent years, the isolation of parasitic DNA from faecal samples and PCR techniques, have been improved and simplified. Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction. These new technical possibilities make it feasible to introduce PCR with its unsurpassed sensitivity and specificity in a routine laboratory setting for the diagnosis of intestinal parasites. Detection rates of the parasitic infections included in the PCR are increased significantly compared with microscopy. Molecular diagnostics, especially in children, reveal a possible cause of the gastrointestinal complaints in many more cases compared with conventional methods. Usually in GP patients no other pathogenic parasites are detected using microscopy and in the returning travellers additional parasites are found with microscopy in a minority of cases only. Multiplex real-time PCR offers a highly sensitive and specific diagnostic alternative for labour intensive microscopy in clinical laboratory practice. Additional diagnostic methods for the detection of parasitic infections that are not included as PCR target can be limited to a selected group of patients.

Immunocytochemical techniques applied to sections and whole-mount preparations of cercariae from two species of trematodes, Trichobilharzia ocellata and Schistosoma mansoni, revealed the occurrence of immunoreactivity (IR) to several neurosubstances in the nervous system (NS). Immunostaining was localized in cerebral ganglia, in the main commissure, in anterior and posterior nerve trunks, as well as in a pair of nerve fibres running along the tail. In T. Ocellata, immunoreactivity (IR) was observed with antisera raised against: glutamate, FMRFamide, catch-relaxing peptide (CARP), small cardiac peptide B (SCPB), arg-vasotocin (AVT), arg-vasopressin (AVP), and substance P. In S. mansoni antisera raised against glutamate, FMRFamide, CARP, SCPB, α-caudodorsal cell peptides (α-CDCP), and cholecystokinin (CCK) showed neuronal IR. With the other 51 antisera tested no IR was observed. With anti-APGWamide, IR was observed outside the NS in cells of the wall of the daughter sporocyst and in flame cells of cercariae of T. ocellata. IR to FMRFamide was present in the escape glands of the intrasporocystic cercariae of T. ocellata and S. mansoni. IR to somatostatin was observed in subtegumental parenchymal cells of cercariae of S. mansoni. IR to met-enkephalin was present in cells of the cercarial embryos and in undifferentiated cells in developing cercariae. Trematodes are, together with cestodes, phylogenetically the oldest classes in which glutamate-like material and immunopositivity to a number of neuropeptides isolated from invertebrates has been demonstrated. The results are discussed in relation to immunocytochemical data obtained for other platyhelminths, to endogenous functions of the immunopositive materials, and to their possible role in parasite–host interactions.

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

The accumulation of the trypanocide isometamidium chloride (Samorin®, RMB Animal Health Ltd, UK) by a range of clones of Trypanosoma congolense with varying sensitivity to the drug, was measured by methods based on the fluorescence of isometamidium. Fluorescence microscopy and flow cytometry showed a reduction in drug accumulation by resistant clones. Fluorescence spectrophotometry demonstrated an inverse correlation between the intensity of cell-associated fluorescence and the level of resistance of the clones expressed in vivo. The addition of the metabolic inhibitor SHAM/glycerol to the incubation medium resulted in a reduction of this apparent difference in drug accumulation between the clones; those clones which were sensitive to isometamidium showed a reduction in fluorescence while a percentage increase in fluorescence was observed as clones became more resistant to the trypanocide. These observations may be of value for the in vitro detection of resistant T. congolense populations and may also be used to estimate the mean level of resistance in a given sample. The results also imply that decreased accumulation of isometamidium by drug-resistant clones of the parasite may be responsible for the reduction in sensitivity.

Ultra-thin sections and electron microscopy have been used in a study of the empty membranes which occur in the fatbody cells of the larva of T. paludosa infected with the Tipula Iridescent Virus (TIV).

It has been observed that these empty membranes are most numerous in the early stages of infection. Later an apparent ‘primary body’, which may be threadlike in its initial stages, develops in the centre of the membrane. This primary body seems to increase in size until the membrane is filled, and the various steps in this process can easily be seen.

It is suggested that the formation of the empty membranes and the apparent gradual development of their contents are the means by which the virus multiplies.

The remarkable resemblance between the empty virus membranes and the endoplasmic reticulum is pointed out.

Single infections of Trichinella spiralis of 48 h duration did not produce a significant degree of immunity in mice.

Single or triple light infections of 7 h duration which, it is suggested, were composed entirely of fourth-stage larvae, did not produce significant immunity.

Single infections of 3 or 4 days duration did produce immunity. It is suggested that the fourth-stage intestinal larvae and the fourth moult are not the most important sources of immunizing antigen in a T. spiralis infection.

I would like to express my gratitude to H.M. Treasury and to the Agricultural Research Council for financial support during the time that this work was being conducted. I would also like to thank Professor W. I. B. Beveridge, within whose department this work was conducted, for the facilities provided. The helpful advice of my colleagues R. M. Connan and R. S. Phillips was of great value during the time that this manuscript was being prepared for publication.

During the early stages of the primary Plasmodium chabaudi chabaudi AS parasitaemia in CBA/Ca mice this parasite invaded normocytes, but as the parasitaemia developed increasing numbers of parasites were seen within reticulocytes. During and just after peak parasitaemia, as further parasite replication was controlled, the ‘crisis’ phase ensued, mice became increasingly anaemic and reticulocyte numbers were markedly increased. As the parasitaemia was resolved during crisis in excess of 25% of parasites had invaded reticulocytes. In phenylhydrazine-pretreated mice with artificially high reticulocyte levels and infected with P. c. chabaudi AS, normocyte/reticulocyte invasion occurred with equal frequency. No reduction in the infectivity of parasite populations developing in reticulocytes was observed.

Two lines of guinea-pigs, whose susceptibilities to infection with the parasitic nematode Trichostrongylus colubriformis differ significantly, have been established. This difference is probably based on genetically determined differences between the ability of members of each line to bring about immune expulsion. of the parasite.

The patterns of association between parasites within a particular host are determined by a number of factors. One of these factors is whether or not infection by one parasite influences the probability of acquiring other parasite species. This study investigates the pattern of association between various parasites of the New Zealand cockle Austrovenus stutchburyi. Hundreds of cockles were collected from one locality within Otago Harbour, New Zealand and examined for trematode metacercariae and other symbionts. Two interspecific associations emerged from the study. First, the presence of the myicolid copepod Pseudomyicola spinosus was positively associated with higher infection intensity by echinostomes. The side-effect of the copepod's activities within the cockle is suggested as the proximate mechanism that facilitates infection by echinostome cercariae, leading to a greater rate of accumulation of metacercariae in cockles harbouring the copepod. Second, a positive association was also found between infection intensity of the metacercariae of foot-encysting echinostomes and that of gymnophallid metacercariae. This supports earlier findings and suggests that the gymnophallid is a hitch-hiker parasite because, in addition to the pattern of positive association, it (a) shares the same transmission route as the echinostomes, and (b) unlike the echinostomes, it is not capable of increasing the host's susceptibility to avian predation. Thus, both active hitch-hiking and incidental facilitation lead to non-random infection patterns in this parasite community.

Cysts of Sarcocystis species were found in 24 of 44 (54·5%) examined blackbirds (Turdus merula). Under the light microscope, only 1 morphological type of cyst was found in all birds investigated. Ribbon-shaped cysts were long (the largest fragment found amounted to 7 mm) and of different thickness (25–206 μm). A cyst wall reached up to 3·5 μm and had finger-like protrusions. Under the transmission electron microscope, a single cyst isolated from 1 blackbird was studied. The cyst wall was 2·5–4·4 μm thick, had club- or irregularly-shaped and sometimes branched protrusions that differed in size. The content of cysts was divided into large chambers by septa. Orange segment-shaped cystozoites were 6·2 × 1·4 (5·5–7·2 × 1·2–1·5) μm. This type of cyst wall has never been described in Sarcocystis species isolated from birds, thus far. The results of 18S rDNA, 28S rDNA and ITS–1 region sequences showed that S. turdusi was most closely related to S. columbae, S. calchasi, S. wobeseri, S. cornixi and Sarcocystis sp. ex Accipiter nisus parasitizing birds. Phylogenetic results suggest that predatory birds are the most probable definitive hosts of S. turdusi.

MicroRNA-132 (miR-132) has been demonstrated to affect multiple neuronal functions and its dysregulation is linked to several neurological disorders. We previously showed that acute Toxoplasma gondii infection induces miR-132 expression both in vitro and in vivo. To investigate the impact of chronic infection on miR-132, we infected mice with T. gondii PRU strain and performed assessment 5 months later in six brain regions (cortex, hypothalamus, striatum, cerebellum, olfactory bulb and hippocampus) by qPCR. We found that while acute infection of T. gondii increases the expression of miR-132, chronic infection has the opposite effect. The effect varied amongst different regions of the brain and presented in a sex-dependent manner, with females exhibiting more susceptibility than males. MiR-132 and brain-derived neurotrophic factor (BDNF, an inducer of miR-132) were not co-varies in the brain areas of infected mice. T. gondii DNA/RNA was found in all tested brain regions and a selective tropism towards the hippocampus, based on bradyzoite density, was observed in both males and females. However, the expressions of miR-132 or BDNF were poorly reflected by the density of T. gondii in brain areas. Our findings highlight the importance of investigating the miR-132-mediated neuronal function in mice infected with T. gondii.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvaII digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.

Species introduced into new areas often show a reduction in parasite and genetic diversity associated to the limited number of founding individuals. In this study, we compared microsatellite and parasite diversity in both native (lower Danube) and introduced populations of 4 Ponto-Caspian gobies, including those (1) introduced from within the same river system (middle Danube; Neogobius kessleri and N. melanostomus), and (2) introduced from a different river system (River Vistula; N. fluviatilis and N. gymnotrachelus). Microsatellite data confirmed the lower Danube as a source population for gobies introduced into the middle Danube. Both native and introduced (same river system) populations of N. kessleri and N. melanostomus had comparable parasite species richness and microsatellite diversity, possibly due to multiple and/or continual migration/introduction of new individuals and the acquisition of local parasites. Reduced parasite species richness and microsatellite diversity were observed in introduced (different river system) populations in the Vistula. A low number of colonists found for N. fluviatilis and N. gymnotrachelus in the Vistula potentially resulted in reduced introduction of parasite species. Insufficient adaptation of the introduced host to local parasite fauna, together with introduction into an historically different drainage system, may also have contributed to the reduced parasite fauna.

An epidemiological cohort study of Enteromyxum scophthalmi in cultured turbot was performed on a farm in North Western Spain. Four different ongrowing stocks (A, B, C, D) were monitored monthly until market size. Fish from stocks C and D were divided into 2 subgroups, receiving filtered (CF and DF) or unfiltered (CUF and DUF) water. The lack of water filtration was positively associated with infection prevalence, as all fish kept in filtered water remained uninfected. Parasite abundance varied seasonally (P<0·05) in stock B and subgroup CUF. Infection was also associated (P<0·05) with host weight, and the highest prevalences and intensities were detected in 101–200 g and 201–300 g fish. Distribution pattern of E. scophthalmi in subgroups CUF and DUF had a variance higher than the mean, indicating overdispersion. The minimum period necessary for the first detection of the parasite and for the appearance of disease symptoms and mortality, varied depending on the stock and introduction date, although a long pre-patent period was always observed. Several factors, such as host density, parasite recruitment and parasite-induced fish mortality can contribute to the observed distribution pattern. Risk factors found to be associated with E. scophthalmi infection, including water quality and accumulation of infective stages in the culture tanks, should be considered when designing control strategies to prevent the introduction and spread of infective stages in the facilities.

The impact of di-cationic pentamidine-analogues against Toxoplama gondii (Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites of T. gondii Rh and T. gondii Me49 with an IC50 of 0·11 and 0·13 μm, respectively. Pre-incubation of fibroblast monolayers with 1 μm DB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5–6 days of drug exposure, T. gondii tachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2 μm. Out of a set of 32 di-cationic compounds screened for in vitro activity against T. gondii, the arylimidamide DB745, exhibiting an IC50 of 0·03 μm and favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adapted T. gondii (IC50=0·07 μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adapted T. gondii tachyzoites, but not on DB750-adapted T. gondii. Adaptation of T. gondii to DB745 (up to a concentration of 0·46 μm) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adapted T. gondii seemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity of T. gondii tachyzoites to adapt to environmental changes, at least under in vitro conditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriate in vivo models.

African trypanosomes have been around for more than 100 million years, and have adapted to survival in a very wide host range. While various indigenous African mammalian host species display a tolerant phenotype towards this parasitic infection, and hence serve as perpetual reservoirs, many commercially important livestock species are highly disease susceptible. When considering humans, they too display a highly sensitive disease progression phenotype for infections with Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, while being intrinsically resistant to infections with other trypanosome species. As extracellular trypanosomes proliferate and live freely in the bloodstream and lymphatics, they are constantly exposed to the immune system. Due to co-evolution, this environment however no longer poses a hostile threat, but has become the niche environment where trypanosomes thrive and obligatory await transmission through the bites of tsetse flies or other haematophagic vectors, ideally without causing severe side infection-associated pathology to their host. Hence, African trypanosomes have acquired various mechanisms to manipulate and control the host immune response, evading effective elimination. Despite the extensive research into trypanosomosis over the past 40 years, many aspects of the anti-parasite immune response remain to be solved and no vaccine is currently available. Here we review the recent work on the different escape mechanisms employed by African Trypanosomes to ensure infection chronicity and transmission potential.