The work reported here describes the comparative ability of albendazole (ABZ), fenbendazole (FBZ) and triclabendazole (TCBZ) to penetrate through the tegument of mature Fasciola hepatica, and the influence of the physicochemical composition of the incubation medium on the drug diffusion process. The data obtained from the trans-tegumental diffusion kinetic studies were complemented with the determination of lipid-to-water partition coefficients (octanol-water) for the benzimidazole (BZD) anthelmintic drugs assayed. Sixteen-week-old F. hepatica were obtained from untreated artificially infected sheep. The flukes were incubated (37 °C) over 60 and 90 min in incubation media (pH 7·4) prepared with different proportions of ovine bile and Krebs' Ringer Tris (KRT) buffer (100, 75, 50, 25 and 0% of bile) containing either ABZ, FBZ or TCBZ at a final concentration of 5 nmol/ml. After the incubation time expired, the liver fluke material was chemically processed and analysed by high performance liquid chromatography (HPLC) to measure drug concentrations within the parasite. Additionally, the octanol-water partition coefficients (PC) for each molecule were calculated (as an indicator of drug lipophilicity) using reversed phase HPLC. The 3 BZD molecules were recovered from F. hepatica at both incubation times in all incubation media assayed. The trans-tegumental diffusion of the most lipophilic molecules ABZ and FBZ (higher PC values) tended to be greater than that observed for TCBZ. Interestingly, the uptake of ABZ by the liver flukes was significantly greater than that measured for TCBZ, the most widely used flukicidal BZD compound. This differential uptake pattern may be a relevant issue to be considered to deal with TCBZ-resistant flukes. Drug concentrations measured within the parasite were lower in the incubations containing the highest bile proportions. The highest total availabilities of the 3 compounds were obtained in liver flukes incubated in the absence of bile. Altogether, these findings demonstrated that the entry of the drug into a target parasite may not only depend on a concentration gradient, the lipophilicity of the molecule and absorption surface, but also on the physicochemical composition of the parasite's surrounding environment.

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3–62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means†.

Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

Understanding the processes that shape parasite diversification, their distribution and abundance provides valuable information on the dynamics and evolution of disease. In this study, we assessed the diversity, distribution, host-specificity and infection patterns of apicomplexan parasites in amphibians and reptiles from Oman, Arabia. Using a quantitative PCR approach we detected three apicomplexan parasites (haemogregarines, lankesterellids and sarcocystids). A total of 13 haemogregarine haplotypes were identified, which fell into four main clades in a phylogenetic framework. Phylogenetic analysis of six new lankesterellid haplotypes revealed that these parasites were distinct from, but phylogenetically related to, known Lankesterella species and might represent new taxa. The percentage of infected hosts (prevalence) and the number of haemogregarines in the blood (parasitaemia) varied significantly between gecko species. We also found significant differences in parasitaemia between haemogregarine parasite lineages (defined by phylogenetic clustering of haplotypes), suggesting differences in host–parasite compatibility between these lineages. For Pristurus rupestris, we found significant differences in haemogregarine prevalence between geographical areas. Our results suggest that host ecology and host relatedness may influence haemogregarine distributions and, more generally, highlight the importance of screening wild hosts from remote regions to provide new insights into parasite diversity.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are vectors of a number of viral diseases worldwide. Following the unforeseen outbreak of bluetongue in northern Europe (2006–2009) there was a need to clarify on-farm breeding substrates utilized by temperate Culicoides spp. Six substrates (cow dung, cow slurry, horse dung, sheep dung, maize silage and soil) were investigated for Culicoides spp. emergence over a 31-week period. Overall, most Obsoletus group Culicoides emerged from the cow dung and the most Pulicaris group Culicoides emerged from the sheep dung. Furthermore, Culicoides of the Obsoletus group were found to be abundant in cow slurry and sheep dung. Temperature played a significant role in the emergence times of adult Culicoides. The Obsoletus group appear to have undergone 3 generations during the experimental period. The sex ratio of emergent Obsoletus group Culicoides was affected by substrate type, with a greater proportion of males emerging from cow dung and slurry compared with the other substrates.

This is the first demonstration of the in vitro development of the 3rd-stage larvae (L3) of Hysterothylacium aduncum to the adult. This was achieved in a semi-defined medium that is easy to prepare and to reproduce. The L3, collected from the peritoneal cavity of horse mackerel (Trachurus trachurus), were individually inoculated into RPMI-1640 medium +20% heat-inactivated fetal bovine serum (IFBS). It has been demonstrated that the optimum temperature for development is around 13 °C and is stimulated by the presence of 5% CO2 in the growth atmosphere, increasing the percentage moulting to the 4th larval stage (L4) by 1.9-fold (from 44 to 82%) and the average survival of the nematodes by 1.6 times (from 60 to 96 days). When the larvae were grown at different pHs, optimum development occurred at pH 4.0. Under these conditions, all the larvae moulted to the L4 and more than two-thirds transformed to the adult stage – in which 25–30% of the females laid eggs – and reached an average survival of over 4 months. When this medium was supplemented with 1% (w/v) of commercial pepsin, all the larvae reached the adult stage, at least 45% of the females oviposited, laying around 12-fold more eggs per female than in the medium without pepsin. The mean size of the eggs (non-fertilized) obtained was 56.8×47.6 μm. The mean length of the adult males obtained was between 3.2 and 5.2 cm and the females were between 3.0 and 6.5 cm. The adult specimens were morphologically identified as Hysterothylacium aduncum aduncum. This culture medium (RPMI-1640+20% (v/v) IFBS+1% commercial pepsin, at pH 4.0, 13 °C and 5% CO2 in air) could facilitate the identification of at least some of the larvae of the genus Hysterothylacium – and perhaps other anisakids – for which the specific identification and the biological study of these parasites is often difficult.

1. The carcasses of 154 silver foxes of various ages, from fifteen farms in the southwest, have been examined and their parasites recorded. In addition, those of fourteen wild red foxes have similarly been examined.

2. The incidence of various species of nematode, cestode, and protozoan parasites has been analysed for the foxes, in age groups and in farm groups.

3. The worm egg counts in the faeces have been discussed in relation to the numbers of female worms in the foxes. No adequate correlation has been established between the two sets of data.

4. The ages of the foxes at incidence of the nematode parasites has been discussed, and the possibility of the development of resistance to various species investigated.

5. Certain peculiarities in the sex ratios of the Nematoda have been discussed.

6. The possibility of infestation of silver foxes by parasites derived from the wild-red-fox population of the neighbourhood of the farms has been investigated, as has also that of the transference of Crenosoma striatum from the hedgehog to foxes.

1. Measurements were made of the head length, head width, total body length, antenna length, length and breadth of 3rd antennal segment of head and body lice from natural infestations. In all measurements (except the last) the means were significantly different for. the two forms, but in all cases there was considerable overlap.

2. Head lice reared continuously on the body for forty-three generations (over 2 years) did not change systematically in size. Body lice reared under similar conditions were likewise constant.

3. There was a highly significant difference between mean measurements of all the samples of head lice reared for up to 2 years on the body when compared with body lice reared for a similar period in the same way. The means for lice worn 24 hr. per day were less than those worn 12 hr. per day in both strains. An experiment showed that this is probably due to a difference in average temperature.

4. The mean live weights of head and body strains were even more distinct than linear dimensions on the several occasions when these were recorded.

5. Measurements of the dimensions of the opercula of the eggs of the two races and the numbers of air cells in them were distributed in the same way as the body measurements: that is, distinct means but overlapping individuals.

6. Ventral abdominal muscles in wild head lice are typically confined to segment 5. (The forty specimens examined adhered to this rule.) In wild body lice, muscles are usually also present in segment 4 as well.

7. Hybrids (female body x male head louse) were fertile for several generations. In size, the F1 generation was intermediate, but the F2 and F3 generations became identical with the body louse strain.

8. Eggs of the body louse strain hatched at the same time as those of the head strain, but the body strain reached a significantly higher percentage hatch.

9. The times of moulting during development were studied in both races in relation to differing opportunities to feed (24, 12, 6 and 3 hr. per day). The two strains showed very similar reactions in regard to speed of development, but throughout there was a higher pre-imaginal mortality in the head strain.

10. Head lice were shown to be considerably more susceptible to starvation than body lice.

11. If adults of the two strains are mixed, there is no tendency towards homogeneous mating. Of the mixed matings, those between body louse males and head louse females are much more common than the reverse.

12. Female head lice show a slight difference in their egg-laying preferences when offered a choice of hair, voile and tape.

My thanks are due to Prof. P. A. Buxton, F.R.S., for a number of helpful suggestions based on his extensive knowledge of louse biology and to Dr J. O. Irwin for critical comments on the statistical issues.

The hamster cheek pouch has been used to investigate the skin migration of schistosomula at the ultrastructural level. Parasites have been observed up to 72 h post-infection. Despite rapidly reaching the epidermal basement membrane schistosomula remain in an epidermal location for at least 40 h post-infection, by which time the acetabular glands and their ducts have been lost and the transformation from cercaria to schistosomulum completed. Entry into the dermis and exit from the skin via the blood vessels is therefore by mechanical means or by lytic secretions emanating from a source other than the acetabular glands. The head gland which persists in the dermal schistosomula is a probable source of such secretions. The observations suggest that the apical area and musculature of the head capsule are important in gaining access to the lumen of blood vessels.

The virulence of Entamoeba histolytica is governed by adhesion/colonization in the gut which is mediated by a galactose specific lectin. Two morphologically identical but distinct species i.e. pathogenic E. histolytica and non-pathogenic E. dispar, can be differentiated by distinct epitopes in the lectin. Both species bind to colonic epithelial cells, but only E. histolytica infection induces an inflammatory response and subsequent pathogenesis. Thus, comparing the responses of the intestinal cells to pathogenic and non-pathogenic lectins is a point of interest. The pathogenic lectin causes cytolysis of epithelial and immune-competent cells. Our data (both qualitative and mRNA quantitation) indicate that the epithelial cells responded to E. histolytica lectin with an increased expression of pro-inflammatory IL-2, IL-6, IL-8, MIP-1α, MCP-1, RANTES, GROα and GMCSF as compared to E. dispar lectin. The pathogenic LCM induced a significant increase in intracellular calcium concentration, proliferative response and chemotaxis of lymphocytes from ALA patients as compared to non-pathogenic LCM. High RANTES and IL-6 were induced in patients' lymphocytes by pathogenic LCM, along with their receptors CCR5 and CD126 as compared to NP-LCM. The local release of such a complex network of cytokines/chemokines could explain the histopathology of E. histolytica infection. The comparative low levels of these chemokines/pro-inflammatory cytokines and high levels of anti-inflammatory IL-10 in response to non-pathogenic E. dispar could explain the absence of an acute inflammatory response and the disease process. The cytokines and chemokines may provide a mechanism for initiation, amplification or containment of inflammation during disease state.

Some parasites with complex life-cycles are able to manipulate the behaviour of their intermediate hosts in a way that increases their transmission to the next host. Gammarids infected by the tapeworm Cyathocephalus truncatus (Cestoda: Spathebothriidea) are known to be more predated by fish than uninfected ones, but potential behavioural manipulation by the parasite has never been investigated. In this study, we tested the hypothesis that C. truncatus is able to manipulate the behaviour of one of its intermediate hosts, Gammarus pulex (Crustacea: Amphipoda). To assess if any behavioural change was linked to other phenotypic alterations, we also measured the immunity of infected and uninfected individuals and investigated the pathogenic effects of the parasite. Infected gammarids were significantly less photophobic than uninfected ones, but no effect of infection on the level of immune defence was found. The results on survival, swimming activity and oxygen consumption suggest that the parasite also has various pathogenic effects. However, the alteration in host phototaxis was not correlated to some of these pathogenic effects. Therefore, we propose that the modification in host reaction to light is a behavioural manipulation, explaining the previously observed increase of gammarid predation rate.

Trichinella spiralis infection causes the transformation of infected muscle cells, which leads to nurse cell formation. To search for the candidate genes responsible for nurse cell formation, cDNA microarray analysis of muscle tissues was performed before and after Trichinella infection. The Atlas mouse 1.2 cDNA expression microarray revealed the expression profiles of 1176 known genes. Out of these, 311 gene expressions were detected in normal and/or infected muscles. After the infection, 184 out of the 311 genes increased in expression by more than 3-fold. These included genes responsible for cell differentiation, proliferation, cell cycle and apoptosis. Thus this study suggested candidate genes for further investigation to dissect the molecular mechanisms of nurse cell formation.

An outbreak of the Microsporidian parasite Thelohania contejeani is reported in a population of Austropotamobius pallipes in Northumberland, U.K. Since less than 10% of all crayfish caught were infected it is concluded that T. contejeani has no serious effect on the population studied. The mode of transmission of the parasite from host to host is discussed.

The host–parasite relationship and life-cycle of T. contejeani were studied using histological and electron microscope techniques. Spores were observed in some detail, and diplocarya, sporont and pansporoblast stages were identified within the general spore mass that invades host muscle tissue. Using serial sections the sporoblast ‘diplocaryon’ was shown to be a single nucleus with a deep cleft. T. contejeani was found to occur mainly in striated muscle, although it was found in cell bodies in the central nervous system.

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to ‘freeze’ gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific ‘sensory’ genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.

In a very important revision of the Streblidae, Speiser (1900) classified them into two subfamilies: (1) the Nycteriboscinae and (2) the Streblinae. To the first subfamily he referred all the genera of the world, with the exception of Strebla and Euctenodes. Upon the presence of the ctenidium and some other characters these two American genera were included in the second subfamily. This classification of the subfamilies was also adopted by Kessel (1924, 1925).

We have isolated and sequenced cDNA clones from Eimeria tenella and Eimeria maxima which encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of the E. tenella calmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells in in vitro culture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells by E. tenella.

A group of thirty-nine Merino-Border Leicester cross-bred lambs were given six sensitizing doses each of 3000 infective Haemonchus contortus larvae. Previous studies showed that this treatment produced a state of immunological exhaustion (Dineen & Wagland, 1966a). In the present study the regeneration of the immunological response was followed by challenging the sensitized animals and previously uninfected control animals with 3000 infective larvae at 2, 4, 8 and 16 weeks after termination of the sensitizing infections with an anthelmintic. Faecal egg counts were carried out on infected animals twice weekly throughout the course of the experiment, and differential worm counts were performed on groups of animals slaughtered 48 days after challenge.

The results show that maximum regeneration of the immune response occurred when periods of 4 and 8 weeks were permitted to elapse between removal of the sensitizing infections and challenge. At 16 weeks the response of the sensitized animals to challenge infection was not significantly different from that of the control animals.

In contrast to results recorded in the previous communication, a high mortality (49%) occurred among the sheep during sensitization. In the earlier experiment the sensitizing infections were given at fortnightly intervals whereas they were given at weekly intervals in the present experiment.

Interpretation of the study is based upon the threshold behaviour of immunological responsiveness in parasite immunity, the occurrence of an immunological latent period and the relationship of this period to the developmental rate of the parasite, the susceptibility of the 4th larval stage to immunological attack, and the immunological privilege of the adult worm. These phenomena have been described in previous communications in the present series.

We are greatly indebted to Mr Emil Teleki and Misses Helen Giller and Lindy Stothart for their excellent technical assistance.

The entomopathogenic nematode Steinernema carpocapsae is mutualistically associated with the bacterium Xenorhabdus nematophila. Infective Juveniles (IJs) transport X. nematophila cells that provide them with good conditions to reproduce within the insect. In the laboratory, long term stationary-phase culture conditions sometimes lead X. nematophila's variant 1 cells, which were previously isolated from the worms, to spontaneously and irreversibly change into a new phenotypic variant (variant 2). In this paper, we tested the ability of each phenotypic variant to (i) be transmitted by IJs, (ii) to optimize the worm's fitness within the insect, and (iii) to counteract the effect of closely related antagonistic bacteria previously shown as being able to totally prevent S. carpocapsae's reproduction within the insect. We found that IJs did associate with cells of both phenotypes but that the variant 2 cells were preferentially retained by the nematodes when both variants were present in the insect. Both phenotypic variants led to the same fitness of S. carpocapsae in insects not infected by antagonistic bacteria. In insects infected by antagonistic bacteria, both variants were able to provide protection to S. carpocapsae. Nevertheless, this protection depended on the phenotypic variant and the antagonistic bacteria that were co-injected into the insect. Further analysis conduced in vitro showed that this variability could be partly linked to the sensitivity of each antagonistic bacterium to xenorhabdicin, produced by X. nematophila.

The transformation of cercariae of Schistosoma mansoni into schistosomula has previously been achieved by passage through membranes of biological origin. This transformation has now been effected in the absence of a membrane. Chemical stimulus for transformation results from contact with human or rat skin lipid and fractionation of the latter led to localization of the active component in a fraction containing phospholipids. The series of processes involved in transformation was subsequently found to be set in motion by the action of egg lecithin-water mixtures on an aqueous suspension of cercariae and yields of 90% of live schistosomula may be obtained reproducibly with crude egg lecithin-water in the ratio 1:50. The juvenile worms were found, by accepted criteria, to be identical to those produced by the normal penetration procedure. Purified lecithin does not produce the same stimulus and the effect of the physical state and of possible impurities present in crude egg lecithin is discussed.

This work was supported by the Conselho Nacional de Pesquisas (Brazil), the Banco Nacional de Desenvolvimento Econâmico FUNTEC contracts nos. 99 and 110 (Brazil), U.S. Army Grants nos. DAHC-19-70-G-0017 (to B.G.) and DAHC-19-70-G-0070 (to J.P.) and by the FORGE Foundation. Contribution no. 18 from the Schistosomiasis Research Unit, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil.

Genomic DNA extracted from entomopathogenic nematodes isolated from 89 soil samples taken throughout the United Kingdom was hybridized with the ribosomal DNA clone from Caenorhabditis elegans (pCe7). When the DNA was digested with EcoR I and Hind III in a double digest, 5 distinct RFLP (restriction fragment length polymorphism) types were observed. While the prevalence of the 5 types was not equal, no correlation with geographical location, soil type or habitat could be detected. Subsequent hybridizations of total genomic DNA from the various RFLP types divided them into 2 groups. The most prevalent group, identified as Steinernema feltiae ( = bibionis), contained 2 of the RFLP types (Al and A2). The other group contained the remaining 3 RFLP types (B1, B2 and B3). Although similar to S. feltiae ( = bibionis), the members of the B-types can be distinguished from this species on morphological grounds and preliminary crossbreeding experiments have demonstrated that the 2 groups are reproductively isolated.