1. Methods are described for the isolation and maintenance of a pure strain of Eimeria mitis.

2. The prepatent period of the infection in chicks was 101 hr. The mean dimensions of the oocysts were 15·38 × 13·83μ, and sporulation occurred after 48 hr. at 60° F. (15·5° C.). The production of oocysts following infection with known numbers of oocysts is recorded.

3. When 500,000 or more oocysts of E. mitis were given to chicks aged 6, 17 or 26 days the growth rate of the birds was markedly reduced. Young chicks were most susceptible and a few deaths occurred when 6-day-old chicks received 2·5 million oocysts. E. mitis is unlikely to be of pathological importance under normal conditions of good husbandry practice.

4. Pathological lesions due to this species of Eimeria were not found in sections of intestinal tissues.

5. Developmental stages of this parasite were found throughout the whole of the small intestine.

6. The growth-retarding effects and oocyst production by E. mitis were controlled when 0·2% sodium sulphadimidine was given in the drinking water.

Resistance to E. necatrix induced by complete life-cycle infections was transferable from duodenum to caeca and vice versa.

Complete life-cycle infections with E. tenella protected against subsequent attempts to infect with 2nd-generation merozoites. A similar experiment using E. necatrix showed a partial effect.

E. necatrix gametogony-only infections did not protect against subsequent gametogony-only infections and did not protect against schizogony in the duodenum. They did suppress oocyst production resulting from entire life-cycle infections.

Satisfactory oocyst production from the intra-caecal inoculation of second generation E. necatrix merozoites was not obtained.

Repeated oral infections with E. tenella or E. necatrix reduced subsequent cross-infections with E. necatrix or E. tenella respectively by approximately half. Both schizogony and gametogony stages were affected.

A similar result was obtained with E. tenella oocyst inoculation when previous infections were initiated by the intra-caecal injection of E. necatrix sporozoites.

Oocyst production from the oral administration of E. tenella oocysts to birds previously infected with gametogony stages only of E. necatrix was much reduced but the schizogony stages of the challenge infection were not appreciably affected. Previous repeated caecal inoculation of E. necatrix merozoites did not reduce oocyst production from subsequent inoculations of E. tenella merozoites or of E. necatrix merozoites.

Thanks are due to Mr P. L. Long for his interest and advice. The excellent technical assistance of Mr Brian Millard and other members of the Parasitology Department is also gratefully acknowledged.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Seven species of marine fishes and three of fresh or brackish water Canadian fishes have yielded new or known Myxosporidia. The marine fishes were from St Andrews, New Brunswick and Halifax, Nova Scotia, places 250–275 miles apart by sea, and from the Labrador coast. The brackish and freshwater fishes were obtained in Montreal and the Salmon River, Nova Scotia.

Descriptions and illustrations are given of the morphology of five new species of Myxosporidia: Ceratomyxa urophysis, Myxoproteus myoxocephali, Myxidium myoxocephali, Myxosoma diaphana and Henneguya amiae.

Chloromyxum leydigi Mingazzini, C. granulosum Davis, Myxidium lieberkuhni Bütschli, Myxidium gadi Georgévitch and Zschokkella hildae Auerbach are recorded from new piscine hosts in Canada.

Some effects of the Myxosporidian infections, both macroscopic and microscopic, on their hosts are considered. Comparison of the effects of similar infections of Myxosporidia on allied fishes in Normandy and Eastern Canada has shown a general similarity between them.

The economic importance of some Myxosporidian infections of the edible fish examined is shown by parasitism of the cod, haddock and sculpin. Amia calva is also used for food in Montreal. The possibility of edible fishes acquiring Myxosporidian infections by way of their food is noted, with reference to the host-specificity of the Myxosporidia of Fundulus heteroclitus described by Bond.

The influence of environment on and powers of adaptation to different habitats exhibited by certain Myxosporidia is discussed.

Some remarks are made on the zoogeographical distribution of certain Myxosporidia and their piscine hosts.

We have isolated a cDNA (SKvl. 1) encoding a Shaker-related K+ channel from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. The deduced amino acid sequence (512 aa, 56·5 kDa) contains 6 putative membrane-spanning domains (S1–S6) and a pore-forming domain (H5). SKv1.1 is grouped in the Shaker family, but forms a unique branch within this family, on the basis of dendrogram analysis. SKv1.1 shows significant sequence identity with most other Shaker channels, with 64—74% identity in the core region (S1–S6). It has the highest sequence identity with the K+ channel (Ak01a) from Aplysia. Northern blot analysis detected a single primary transcript of 2·8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. Expression of SKv1. 1 in Xenopus oocytes produced a rapidly activating and inactivating outward K+ current which is highly sensitive to 4-aminopyridine, but is insensitive to tetraethylammonium, mast cell degranulating peptide, dendrotoxin and charybdo-toxin. The presence of a Shaker homologue in Schistosoma suggests that Sh subfamilies may exist in other lower invertebrates as well as platyhelminths.

The fine structure of the nervous system in the scolex and neck region of Hymenolepis nana has been investigated by transmission electron microscopy. A description of the gross neuroanatomy in these regions of the worm is presented. The ganglia, commissures and nerve cords consist of an incomplete cortex of nerve cell bodies, and a core of nerve fibres. A delimiting sheath or capsule is absent. The nerve cell bodies contain a single nucleus with a single nucleolus, mitochondria, many ribosomes, Golgi complexes and vesicles formed within the Golgi cisternae. Numerous sub-surface cisternae are present beneath the outer plasma membrane of the nerve cell body, and the inner surfaces of these cisternae are studded with ribosomes. Some of the cisternae run tangentially into the cytoplasm of the perikaryon, particularly in the vicinity of the Golgi complexes; both sides of these cisternae are studded with ribosomes. From each neuronal perikaryon arise one or more neurites that contain neurotubules, mitochondria, ribosomes and electron-lucent or dense-cored vesicles. Five types of vesicle have been distinguished on the basis of their size and content. The neurites are unmyelinated and form synapses in the neuropile; the synapses possess features typical of those where mechanical strength is of importance. Three types of sensory receptors have been observed in H. nana, two ciliated and one non-ciliated; the latter typically form double or triple nerve endings which terminate within the tegument.

The response of sheep given six sensitizing infections each of 3000 infective larvae of H. contortus at fortnightly intervals to a challenge infection of 3000 infective larvae has been studied. While a large proportion of the challenge infection (ca. 3000 parasites) persisted to termination of the experiment on day 141 in sheep (group 3) in which the challenge was superimposed upon the sensitizing infections, only a mean of 437 worms, including both 4th-stage larvae and adults, was recorded at this time in another group (group 5) in which the sensitizing infections were terminated by anthelmintic treatment on day 90, i.e. 8 days before challenge. The proportion of the total worm burdens of the challenge infections arrested at the 4th larval stage was increased for both groups (25% for group 3 and 18% for group 5) by comparison with unsensitized control groups (4% for group 6 and 2% for group 7).

As the effect of immunological control was expressed more vigorously in the group (group 5) in which the sensitizing infections were terminated before challenge it seems likely that recovery of immunological competence occurred in these animals following removal of the sensitizing infections. This conclusion suggests that, owing to the persistence of the sensitizing infections and therefore exposure of the immune mechanism to continuous antigenic insult, the animals which received the superimposed challenge infection had entered a phase of immunological exhaustion during the challenge period.

Soil-transmitted helminthiasis (STH), among the most common neglected tropical diseases, is a major public health problem in Malaysia with a possible impact on the nutritional status and school participation of rural children. This study was carried out among Aboriginal schoolchildren, living in an endemic area for STH in Malaysia, to determine the possible relationship between intestinal helminthiasis and school absenteeism. We also evaluated whether successful treatment of the infection will affect school attendance among the subjects. Stool analysis revealed that more than 90% of the subjects were infected with at least 1 helminth species, with Ascaris lumbricoides and Trichuris trichiura infections being most prevalent. Infection of moderate-to-heavy worm burdens, low level of fathers' education and anaemia were identified as the significant predictors of high absenteeism among the subjects (P<0·05). Following treatment of the infected children, it was found that school absenteeism was reduced significantly (P<0·01). In conclusion, STH continues to have significant impacts on public health, particularly in rural communities with a negatively significant effect on the school participation of Aboriginal children. A school-based de-worming programme should be introduced and incorporated in the current educational assistance targeted towards the Aboriginal communities, under the auspices of the government.

Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites.

Infections with Theileria parva in the African buffalo are invariably asymptomatic, whereas infections in cattle usually result in clinical disease, the severity of which varies in different populations of cattle. The parasite exhibits antigenic heterogeneity, which in cattle manifests as differences between parasite strains in their cross-protective properties. A series of studies on T cell responses to T. parva in cattle have demonstrated that class I MHC-restricted cytotoxic T lymphocytes (CTL), specific for parasitized lymphoblasts, are important mediators of immunity. Cytotoxic T cell responses frequently display parasite strain-restricted specificities which appear to correlate with the capacity of strains to cross-protect. The strain specificity of CTL responses varies in animals immunized with the same parasite strain and is influenced by both host and parasite genotype. Recent studies have provided evidence that there is competition between epitopes for induction of CTL responses, which can result in a bias to strain-specific epitopes. These properties of the CTL response have important implications for vaccination. Thus, in designing a vaccine, it may be possible, by selecting parasite proteins containing appropriate CTL epitopes, to generate CTL responses that protect against a wide range of parasite strains. Although there are no comparable data on CTL responses in the buffalo, it is considered that the features of the immune response described for cattle would be advantageous for survival of parasite populations in the buffalo. Specifically, a bias in the immune responses to strain-specific determinants should favour establishment of infection in buffalo already carrying the parasite and allow fluctuation in the levels of different parasite strains during the course of persistent infection.

Bat flies (Streblidae) are diverse, obligate blood-feeding insects and probably the most conspicuous ectoparasites of bats. They show preferences for specific body regions on their host bat, which are reflected in behavioural characteristics. In this study, we corroborate the categorization of bat flies into three ecomorphological groups, focusing only on differences in hind leg morphology. As no detailed phylogeny of bat flies is available, it remains uncertain whether these morphological differences reflect the evolutionary history of bat flies or show convergent adaptations for the host habitat type. We show that the division of the host bat into three distinct habitats contributes to the avoidance of interspecific competition of bat fly species. Finally, we found evidence for density-dependent competition between species belonging to the same ecomorphological group.

Amblyomma birmitum sp. nov. is formally described as a new record from 99 Ma old Burmese amber from Myanmar. This confirms the presence of the extant hard tick genus Amblyomma C.L. Koch, 1844 (Ixodida: Ixodidae) in the Late Cretaceous. This discovery is placed in its wider context and some reports of fossil hard ticks, such as a Hyalomma C.L. Koch, 1844 in Eocene Baltic amber, are misidentifications. The genus Amblyomma belongs to the clade Metastriata, a group which probably also accommodates two extinct genera, Cornupalpatum Poinar and Brown, 2003 and Compluriscutata Poinar and Buckley, 2008, also found in Burmese amber. All three fossils are thus only a little younger than published molecular divergence time estimates (ca. 124 ± 17 Ma) for the Metastriata lineage. Amblyomma has a largely Gondwanan distribution today. However, in some biogeographical scenarios, e.g. the Samafrica model, its predicted radiation time postdates the dissolution of the original Gondwana supercontinent raising questions about how its current distribution pattern was achieved.

Human strongyloidiasis is a serious disease mostly attributable to Strongyloides stercoralis and to a lesser extent Strongyloides fuelleborni, a parasite mainly of non-human primates. The role of animals as reservoirs of human-infecting Strongyloides is ill-defined, and whether dogs are a source of human infection is debated. Published multi-locus sequence typing (MLST) studies attempt to elucidate relationships between Strongyloides genotypes, hosts, and distributions, but typically examine relatively few worms, making it difficult to identify population-level trends. Combining MLST data from multiple studies is often impractical because they examine different combinations of loci, eliminating phylogeny as a means of examining these data collectively unless hundreds of specimens are excluded. A recently-described machine learning approach that facilitates clustering of MLST data may offer a solution, even for datasets that include specimens sequenced at different combinations of loci. By clustering various MLST datasets as one using this procedure, we sought to uncover associations among genotype, geography, and hosts that remained elusive when examining datasets individually. Multiple datasets comprising hundreds of S. stercoralis and S. fuelleborni individuals were combined and clustered. Our results suggest that the commonly proposed ‘two lineage’ population structure of S. stercoralis (where lineage A infects humans and dogs, lineage B only dogs) is an over-simplification. Instead, S. stercoralis seemingly represents a species complex, including two distinct populations over-represented in dogs, and other populations vastly more common in humans. A distinction between African and Asian S. fuelleborni is also supported here, emphasizing the need for further resolving these taxonomic relationships through modern investigations.

Complex life cycles are common in free-living and parasitic organisms alike. The adaptive decoupling hypothesis postulates that separate life cycle stages have a degree of developmental and genetic autonomy, allowing them to be independently optimized for dissimilar, competing tasks. That is, complex life cycles evolved to facilitate functional specialization. Here, I review the connections between the different stages in parasite life cycles. I first examine evolutionary connections between life stages, such as the genetic coupling of parasite performance in consecutive hosts, the interspecific correlations between traits expressed in different hosts, and the developmental and functional obstacles to stage loss. Then, I evaluate how environmental factors link life stages through carryover effects, where stressful larval conditions impact parasites even after transmission to a new host. There is evidence for both autonomy and integration across stages, so the relevant question becomes how integrated are parasite life cycles and through what mechanisms? By highlighting how genetics, development, selection and the environment can lead to interdependencies among successive life stages, I wish to promote a holistic approach to studying complex life cycle parasites and emphasize that what happens in one stage is potentially highly relevant for later stages.

Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

The lectin-inhibitory sugars D-glucosamine (GlcN) and N-acetyl D-glucosamine (GlcNAc) are known to enhance susceptibility of the tsetse fly vector to infection with Trypanosoma brucei. GlcNAc also stimulates trypanosome growth in vitro in the absence of any factor derived from the fly. Here, we show that GlcNAc cannot be used as a direct energy source, nor is it internalized by trypanosomes. It does, however, inhibit glucose uptake by binding to the hexose transporter. Deprivation of D-glucose leads to a switch from a metabolism based predominantly on substrate level phosphorylation of D-glucose to a more efficient one based mainly on oxidative phosphorylation using L-proline. Procyclic form trypanosomes grow faster and to higher density in D-glucose-depleted medium than in D-glucose-rich medium. The ability of trypanosomes to use L-proline as an energy source can be regulated depending upon the availability of D-glucose and here we show that this regulation is a graded response to D-glucose availability and determined by the overall metabolic state of the cell. It appears, therefore, that the growth stimulatory effect of GlcNAc in vitro relates to the switch from D-glucose to L-proline metabolism. In tsetse flies, however, it seems probable that the effect of GlcNAc is independent of this switch as pre-adaptation to growth in proline had no effect on tsetse infection rate.

The nervous system of young and adult Amphilina foliacea was studied with immunocytochemical, electron microscopical and spectrofluorometrical methods. The general neuroanatomy is described in detail. New data on the structure and development of the brain were obtained. The 5-HT and GYIRFamide-immunoreactivities occur in separate sets of neurones. The innervation of the reproductive organs is described. The fine structure of 2 types of neurones in the CNS, a sensory neurone, a ‘glial’ cell type, the neuropile and the synapses are described. The level of 5-HT varies between 0·074 and 0·461 μg/g wet weight. This is the first detailed study of the nervous system of A. foliacea. Earlier data on the structure of the nervous system in A. foliacea published in Russian are introduced into the discussion. The study provides data that can be used when considering the phylogenetic position of Amphilinidea.

Concentrations of the elements As, Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, Sn, V, Zn were analysed by inductively coupled plasma mass spectrometry (ICP-MS) in the acanthocephalan Pomphorhynchus laevis and its fish host Barbus barbus. A total of 27 barbels were collected from the Danube River in autumn 2006 close to the town Kozloduy (685 river kilometer) on the Bulgarian river bank. Fish were divided into 3 groups. According to their P. laevis infrapopulation size hosts were considered as heavily infected (>100 worms per fish) and lightly infected (<20 worms per fish). The third group was used to compare heavy metal concentrations between male and female P. laevis. The 5 elements As, Cd, Cu, Pb and Zn were detected in significantly higher concentrations in parasites compared to host tissues (muscle, intestine, liver). According to the calculated mean bioconcentration factors, 3 more elements (Co, Mn, V) showed usually higher concentrations in P. laevis. Comparisons between heavily and lightly infected fish revealed significant differences only for V with higher concentrations for the heavily infected group. Concerning sex-specific metal accumulation V and Zn showed significant differences (V, at P<0·05; Zn, at P=0·05), with higher levels of both metals in females of P. laevis. Our results suggest that – for the metals analysed – the size of the parasite infrapopulation plays no role in the degree of metal accumulation. Similarly, parasite sex seems not to be a crucial factor for metal accumulation in the parasites. Thus, for metal monitoring purposes there is no need to take these aspects into account, which makes the use of parasites as bioindicators more applicable.

The nematode parasite, Nippostrongylus brasiliensis, induces a biphasic anorexia in its rat host. The mechanisms, underlying this anorexia and its possible advantages to the host or parasite are unknown. We have investigated the effect of acute (12–24 h) and chronic (2–17 days) infections on plasma concentrations of leptin, insulin and corticosterone, and on hypothalamic expression of neuropeptide Y, galanin and corticotrophin-releasing factor genes. Plasma leptin was elevated in infected rats relative to uninfected ad libitum-fed controls and pair-fed controls in 12 h infections initiated at dark onset and in infections of 2 days' duration. At other times prior to parasite expulsion, plasma leptin in infected and pair-fed rats was lower than that of uninfected ad libitum-fed controls, reflecting the existing state of negative energy balance. Elevated plasma leptin concentrations in infected rats at day 2 post-infection were accompanied by reduced neuropeptide Y gene expression in the hypothalamic arcuate nucleus compared with both ad libitum control and pair-fed animals, and by lowered corticotrophin-releasing factor gene expression in the paraventricular nucleus relative to pair-feds. Twelve hour infections were characterized by a substantial increase in plasma corticosterone that was independent of reduced food intake, and in 12 h infections initiated at dark onset, where plasma leptin was elevated, there was also increased plasma insulin concentration in infected rats. In longer infections, differences between the groups in plasma insulin and corticosterone concentration were only observed at day 4 post-infection. In summary, perturbations to leptin, insulin and corticosterone signals early in infection may have a causative role and might feed back onto hypothalamic gene expression, whereas subsequent changes in these parameters are more likely to be secondary to negative energy balance.

Intact, viable adults, infective and newborn larvae of Trichinella pseudospiralis were surface labelled with 125I by the chloramine T method and labelled proteins were compared with those obtained from equivalent stages of T. spiralis. Electron-microscope autoradiography determined that labelled proteins were restricted to the cuticle for all stages of both isolates. Comparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS–PAGE), using thin gradient gel slabs, of proteins obtained from each stage, demonstrated that the profile of surface-labelled proteins of T. pseudospiralis were restricted in number, stage specific, and similar to equivalent proteins of T. spiralis both in size and in their organization into aggregates. The stage-specific profiles of surface-labelled proteins derived from newborn larvae were indistinguishable, but differences were noted between adults and infective larvae of the two isolates. These differences in protein structure were confirmed by two dimensional mapping of tryptic peptides. Stage-specific profiles were also obtained when [35S]methionine biosynthetically labelled secretions of the 3 stages of T. pseudospiralis were compared by SDS–PAGE. Comparison of the profiles obtained with secretions for respective stages of T. spiralis again failed to distinguish newborn larvae, but adults and infective larvae of T. spiralis and T. pseudospiralis displayed a mixture of common and species-specific proteins. These findings are discussed in relation to the different pathology associated with infection with two isolates.