This study set out to determine whether the sex ratio of fleas collected from host bodies is a reliable indicator of sex ratio in the entire flea population. To answer this question, previously published data on 18 flea species was used and it was tested to see whether a correlation exists between the sex ratio of fleas collected from host bodies and the sex ratio of fleas collected from host burrows. Across species, the female:male ratio of fleas on hosts correlated strongly with the female:male ratio of fleas in their burrows, with the slope of the regression overlapping 1. Controlling for flea phylogeny by independent contrasts produced similar results. It was also ascertained whether a host individual is a proportional random sampler of male and female fleas and whether the sex ratio in flea infrapopulations depends on the size of infrapopulations and on the gender and age of a host. Using field data, the sex ratio in infrapopulations of 7 flea species parasitic on 4 rodent species was analysed. Populations of 3 species (Nosopsyllus iranus, Parapulex chephrenis and Xenopsylla conformis) were significantly female-biased, whereas male bias was found in 1 species (Synosternus cleopatrae). In general, the sex ratio of fleas collected from an individual rodent did not differ significantly from the sex ratio in the entire flea population. Neither host gender, and age nor number of fleas co-occurring on a host affected (a) the sex ratio in flea infrapopulations and (b) the probability of an infrapopulation to be either female- or male-biased.

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

The gut epithelial barrier is a strategic place to prevent, or at least to limit, parasite dissemination upon oral infection with Toxoplasma gondii. Innate immunity to this pathogen results from delicate interactions involving different components of the infecting agent and the host. We herein aimed to examine the molecular mechanism by which protozoan DNA boosts the production of α-defensin-5 (DEFA-5), the main antimicrobial peptide at the target site of infection. The present study shows that DEFA-5 is rapidly upregulated in intestinal epithelial cells following intracellular Toll-like receptor 9 (TLR9) activation by unmethylated CpG motifs in DNA from T. gondii (CpG-DNA). Concomitantly, CpG-DNA purified from the pathogen markedly increased TLR9 mRNA expression levels in the Caco-2 cell line. We further verified that DEFA-5 production was dependent on interferon-β released from these cells upon treatment with CpG-DNA prepared from tachyzoites. Our results suggest that, in protozoan DNA-stimulated intestinal epithelial cells, the TLR9/interferon-β/DEFA-5 pathway may initiate an innate anti-T. gondii response without the need of parasite invasion. These findings highlight the key role of the gut epithelium in Toxoplasma recognition and amplification of local host defence against this microbe, thereby contributing to gain insight into immunoprotective mechanisms and to improve therapeutic strategies.

The paper describes the development and analysis of a mathematical framework for the study of the within-host population dynamics of the interaction between macroparasites and the human immune system. Simple models of this interaction based on the proliferation of T cell clones specific to parasite antigen, and the impact of clonal expansion on parasite survival, capture the basic features of age-related changes in worm loads within human communities. The model is generalized to multiple epitopes on a single antigen, and reveals competitive exclusion amongst T cells, with a single clone becoming immunodominant in the absence of cross-reactive responses and genetic variation. The introduction of genetic heterogeneity and concomitant variability in the immunogenicity of specific epitopes induces additional complexity into the dynamical interaction. Most importantly, multiple epitope models with antigenic variation suggest that the immunodominant response may not necessarily be targeted at the epitope at which some strains show the greatest immunogenicity. High immunogenicity at a particular epitope can be masked by genetic variability even though many of the variants are more immunogenic at this epitope by comparison with the epitope to which the immunodominant immunological response is directed.

Trypanocide resistance remains a huge challenge in the management of animal African trypanosomiasis. Paucity of data on the prevalence of multi-drug resistant trypanosomes has greatly hindered optimal veterinary management practices. We use mathematical model predictions to highlight appropriate drug regimens that impede trypanocide resistance development in cattle. We demonstrate that using drugs in decreasing resistance order results in a negligible increase in number of cattle with resistant infection, in contrast to a more pronounced increase from trypanocide use in increasing resistance order. We demonstrate that the lowest levels of trypanocide resistance are achieved with combination therapy. We also show that increasing the number of cattle treated leads to a progressive reduction in the number of cattle with drug resistant infections for treatments of up to 80% of the cattle population for the combination treatment strategy. Our findings provide an initial evidence-based framework on some essential practices that promote optimal use of the handful of trypanocides. We anticipate that our modest forecasts will improve therapeutic outcomes by appropriately informing on the best choice, and combination of drugs that minimize treatment failure rates.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (<0.01 larvae per mg tissue), and 5/35 (14.3%) were equivocal. To evaluate sensitivity, a partial ITS1 gene was cloned, and serial plasmid dilutions were created ranging from 100 copies μL−1 to ~1 copy μL−1. All three assays consistently detected 50–100 copies μL−1 in triplicate and qPCR was able to detect ~13 copies μL−1 in triplicate. RPA-EXO was able to detect 25 copies μL−1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL−1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Neuroinvasive larvae of the common dog and cat roundworms, Toxocara canis and Toxocara cati, may cause severe neurological and neuropsychological disturbances in humans. Despite their pathogenic potential and high prevalence worldwide, little is known about their cell-specific influences and cerebral host–pathogen interactions in neurotoxocarosis. To address this discrepancy, a co-culture system of viable larvae with murine neuronal (CAD), oligodendrocytal (BO-1) and microglial (BV-2) cell lines has been established. Additionally, murine adult brain slices have been co-cultured with Toxocara larvae to consider complex organotypic cell–cell interplay. Cytotoxicity of larval presence was measured enzymatically and microscopically. Microscopic evaluation using trypan blue exclusion assay revealed to be less reliable and sensitive than the lactate dehydrogenase activity assay. Ultimately, even low numbers of both T. canis and T. cati larvae have impaired survival of differentiated CAD cells, which morphologically resemble primary neurons. In contrast, viability of oligodendrocytal and microglial cells as well as brain slices was not impaired by larval presence. Therefore, immune-mediated mechanisms or trauma by migrating larvae presumably induce the in vivo pathology rather than acute cytotoxic effects. Conclusively, the helminthic larvae co-culture system presented here is a valuable in vitro tool to study cell-specific effects of parasitic larvae and their products.

Manipulative parasites with complex life cycles are known to induce behavioural and physiological changes in their intermediate hosts. Cyathocephalus truncatus is a manipulative parasite which infects Gammarus pulex as intermediate host. G. pulex males display pre-copulatory mate guarding as a response to male-male competition for access to receptive females. In this paper, we tested the influence that C. truncatus-infection might have on male G. pulex sperm number and pairing success. We considered 3 classes of G. pulex males in our experiments: (i) uninfected males found paired in the field, (ii) uninfected males found unpaired in the field, or (iii) infected males found unpaired in the field. Both infected males and uninfected unpaired males paired less with a new female than uninfected paired males did. Furthermore, infected males appear to be at a strong disadvantage when directly competing for females with a healthy rival male, and had fewer sperm in their testes. We discuss the potential effect of male and female mating strategies on such male host mating alteration.

Thanks to modern molecular biology methods, our understanding of the impact of (endo)symbiotic bacteria on parasitic protists and helminths is growing fast. In this issue, 9 papers have been brought together that describe various facets of the relationships between these microorganisms, reveal their range and high frequency, as well as their capacity to create novel biological complexity. Comparative analyses of these host–endosymbiont interactions indicate that there may be no discrete types of relationships but rather a continuum ranging from a dispensable endosymbiont minimally integrated within the host cell to organelles, such as mitochondria and plastids that evolved into an indispensable, deeply integrated components of the cell. We hope that this series of studies on parasites and (endo)symbiotic bacteria will increase awareness about these relationships and their representation in microbial ecology models.

In hybridogenetic systems, hybrid individuals are fully heterozygous because one of the parental genomes is discarded from the germinal line before meiosis. Such systems offer the opportunity to investigate the influence of heterozygosity on susceptibility to parasites. We studied the intensity of lung parasites (the roundworm Rhabdias bufomis and the fluke Haplometra cylindracea) in 3 populations of water frogs of the Rana lessonae-esculenta complex in eastern France. In these mixed populations, hybrid frogs (R. esculenta) outnumbered parental ones (R. lessonae). Despite variation in parasite intensity and demographic variability among populations, the relationship between host age and intensity of parasitism suggests a higher susceptibility in parentals than in hybrids. Mortality is probably enhanced by lung parasites in parental frogs. On the other hand, while parental frogs harboured higher numbers of H. cylindracea than hybrid frogs, the latter had higher numbers of R. bufonis. Despite such discrepancies, these results support the hybrid resistance hypothesis, although other factors, such as differences in body size, age-related immunity, differential exposure risks and hemiclonal selection, could also contribute to the observed patterns of infection.

Neck samples from 54 badgers and 32 tongue samples of the same badgers (Meles meles), collected in the Lothians and Borders regions of Scotland, were tested using polymerase chain reactions (PCRs) directed against the 18S ribosomal DNA and the internal transcribed spacer (ITS1) region of protozoan parasites of the family Sarcocystidae. Positive results were obtained from 36/54 (67%) neck and 24/32 (75%) tongue samples using an 18S rDNA PCR. A 468 base pair consensus sequence that was generated from the 18S rDNA PCR amplicons (KX229728) showed 100% identity to Sarcocystis lutrae. The ITS1 PCR results revealed that 12/20 (60%) neck and 10/20 (50%) tongue samples were positive for Sarcocystidae DNA. A 1074 bp consensus sequence was generated from the ITS1 PCR amplicons (KX431307) and showed 100% identity to S. lutrae. Multiple sequence alignments and phylogenetic analysis support the finding that the rDNA found in badgers is identical to that of S. lutrae. This parasite has not been previously reported in badgers or in the UK. Sarcocystis lutrae has previously only been detected in tongue, skeletal muscle and diaphragm samples of the Eurasian otter (Lutra lutra) in Norway and potentially in the Arctic fox (Vulpes lagopus).

We investigated the prevalence of sarcocystosis in 826 goats slaughtered in the winter season from November to April in northern Iraq. The prevalence of macrocysts was on average 34%, with only 20% infected animals in November, but 46% in February. The infection rate in 1-, 3- and 6-year-old goats was 4%, 48%, and 83%, respectively. The highest specificity of infection was in the oesophagus (99%) and the lowest in the diaphragm (3%). Grossly, we identified 2 forms of macroscopic sarcocysts, fat and thin, with different morphological characteristics. The prevalence of microcysts was 97% and no effects of age, sex and seasonal variations were observed. Development of microcysts in the small intestine of dogs and cats has also been investigated. The pre-patent period in experimentally infected dogs was 12–14 days and the patent period lasted 64–66 days. A dog shed about 155 million sporocysts, but no sporocysts were shed by cats that had been fed the same infected tissues, thus identifying the microcysts as Sarcocystis capracanis.

A study was made of the protective effects of plasma (CMP) from mice convalescent from infection with Trypanosoma cruzi. A single dose of CMP was injected into mice infected with blood trypomastigotes of 1 of each of 5 strains of T. cruzi. Protection was greatest with strains BG, M1 and Y, and least with strain Peru. Strain Tulahuen was of intermediate susceptibility. The protective effect of CMP was found to be similar in mice infected by metacyclic trypomastigotes harvested from vector bugs and mice infected by blood trypomastigotes. Plasma (IMP) from mice hyperimmunized with 6 doses of a killed T. cruzi epimastigote vaccine with saponin as an adjuvant gave no protection against challenge with strain Y, although a group of mice hyperimmunized in parallel with those from which IMP was taken were strongly resistant to challenge.

An ultrastructural examination of Fellodistomum fellis has revealed that the caecal lining consists of 2 distinct components: (a) cup-shaped digestive cells and (b) a pleomorphic layer of cytoplasm which supports and separates individual digestive cells. The digestive cells sequester host blood components within apical pockets formed by lamellated extensions of the cell surface, and undergo asynchronous, cyclical transformations in morphology associated with extracellular digestion and with the extrusion to the gut lumen of pigmented digestive residues. Histochemical tests and elemental analysis of the pigment suggest that it is a ferripor-phyrin, haematin. In the anterior portion of the caecum the supporting cytoplasmic layer is in continuity with the oesophageal tegument and snares the same ultrastructure. It is concluded that the digestive cells are supported by an extension of the foregut tegument.

Infection by the cestode Taenia taeniaeformis was investigated within numerous cyclic populations of the fossorial water vole Arvicola terrestris sampled during 4 years in Franche-Comté (France). The relative influence of different rodent demographic parameters on the presence of this cestode was assessed by considering (1) the demographic phase of the cycle; (2) density at the local geographical scale (<0·1 km2); (3) mean density at a larger scale (>10 km2). The local scale corresponded to the rodent population (intermediate host), while the large scale corresponded to the definitive host population (wild and feral cats). General linear models based on analyses of 1804 voles revealed the importance of local density but also of year, rodent age, season and interactions between year and season and between age and season. Prevalence was significantly higher in low vole densities than during local outbreaks. By contrast, the large geographical scale density and the demographic phase had less influence on infection by the cestode. The potential impacts of the cestode on the fitness of the host were assessed and infection had no effect on the host body mass, litter size or sexual activity of voles.

There is currently no information on the genome size and complexity in the entomoparasitic nematodes Steinernema and Heterorhabditis. DNA reassociation kinetics were used to determine the genome size and complexity in 2 species: Steinernema carpocapsae and Heterorhabditis bacteriophora. C0t curves derived from renaturation kinetics of denatured DNA indicate that the genomes of these entomoparasitic rhabditids are different both in size and complexity. Genome sizes were estimated at 2·3 × 108 bp for S. carpocapsae and 3·9 × 107 bp for H. bacteriophora and repetitive DNA contents were found to represent 39% and 51% of these respective genomes.

A deterministic mathematical model which predicts the probability of developing a new drug-resistant parasite population within the human host is reported. The model incorporates the host's specific antibody response to PfEMP1, and also investigates the influence of chemotherapy on the probability of developing a viable drug-resistant parasite population within the host. Results indicate that early treatment, and a high antibody threshold coupled with a long lag time between antibody stimulation and activity, are risk factors which increase the likelihood of developing a viable drug-resistant parasite population. High parasite mutation rates and fast PfEMP1 var gene switching are also identified as risk factors. The model output allows the relative importance of the various risk factors as well as the relationships between them to be established, thereby increasing the understanding of the conditions which favour the development of a new drug-resistant parasite population.

A study was carried out to determine if snail nutrition influences infectivity of Schistosoma mansoni cercariae for mice. The percentage of worms recovered after infection was the chief criterion used to determine infectivity.Six groups of Biomphalaria glabrata were raised on separate diets of (1) a standard formula with and without lettuce, (2) ‘Gaines Meal’ dog food (compressed pellets), (3) a commercial chicken feed, (4) Romaine lettuce, (5) squash, and (6) the rhizome of a Caladium plant. At 6 weeks the snails were infected and the cercariae resulting from the different snail infections were used to infect mice. Seven weeks later the mice were sacrificed and worms were recovered by perfusion.Snails raised on squash, lettuce and the Caladium rhizome were only one-half the size of those receiving either the standard formula or the ‘Gaines Meal’ at the time of infection, and produced fewer than one-fifth as many egg clutches. However, the percentage parasitemia on all six groups of mice only varied from 49 to 55%, and there were no apparent differences in the size or development of the worms from the different groups of mice.