Biological characteristics of infectivity, growth rate and fecundity of Hymenolepis diminuta isolated from wild Rattus rattus in Japan were compared with parasites of Texas origin maintained for several generations in this and many other laboratories in laboratory bred Rattus norvegicus. The timing of development and maturation was similar in parasites from both sources, but the mean parasite dry weight was less and the mean egg production lower in Japanese parasites in both single and multiple infections. The differences persisted over 10 weeks in single infections, and were unaffected by rat strain. In all experiments there was much greater variation and heterogeneity in the biological characteristics of the Japanese parasites. It was concluded that the Texas parasites were better adapted to R. norvegicus and in the course of adaptation had become more homogeneous, and that the difference between the parasite strains had a complex genetic basis. Some Japanese parasites of the early isolated generations failed to produce normal eggs, but the proportion of these decreased in later generations. However, selected individual Japanese parasites whose egg production was equal to that of parasites of Texas origin did not produce offspring of larger mean size or higher mean fecundity, and heterogeneity of these characteristics was maintained. In the intermediate host, the mean number of cysticercoids of the Japanese isolate per beetle was higher than that of the Texas strain in Tribolium confusum but lower in Tenebrio molitor. The greater heterogeneity exhibited by Japanese parasites and the genetic basis of the strain difference are discussed with particular reference to geographical isolation, differences in definitive hosts and co-evolution of wild and laboratory host and parasite populations.

Glutathione transferases (GSTs) are a family of multifunctional enzymes with fundamental roles in cellular detoxication. Here we report the molecular characterization of 3 recombinant GSTs belonging to the mu- and delta-class from the parasitic mite Sarcoptes scabiei. Kinetic constants were determined, and the effect of acaricides, including organothiophosphates, pyrethroid esters, a formamidine, a macrocyclic lactone, an organochlorine as well as a bridged diphenyl acaricide, on the activity of the GSTs were tested using 1-chloro-2,4-dinitrobenzene (CDNB) as model substrate. Our results showed that enzymes from the same class and with high amino acid sequence identity have significantly different kinetic properties. For instance, one mu-class GST lost more than 50% of its activity in the presence of one of the organothiophosphates while the activity of the second mu-class GST was only slightly reduced under identical conditions. Tertiary structure modulations indicated that structural differences were the crucial factor for the different kinetic patterns observed. Genome analysis showed that the two mu-class GSTs are organized in tandem in the S. scabiei genome. Taken together these results show that GSTs might be involved in the metabolism of acaricides in S. scabiei.

Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-type N-glycans on the NEJ tegument may have potential for disrupting infection.

Burrowing depth may affect predation rate, feeding ability and reproduction in bivalve clams. We studied the effect of burrowing depth on the abundance of the ergasilid Paraergasilus rylovi in the freshwater bivalve clam Anodonta piscinalis. We transplanted uninfected clams to a lake where they were allowed to choose their preferred burrowing depth, and were exposed naturally to copepodids of the parasite. There was a significant positive correlation between proportionate burrowing depth (PBD) and the abundance of P. rylovi at the end of the 17-day experiment, the deeper-burrowed clams harbouring more P. rylovi. Original PBD (0%, 50%, 100%) did not influence the final PBD or parasite abundance. Clam length affected PBD, smaller clams burrowing deeper, but it did not affect parasite abundance. Infected experimental clams and naturally-burrowed uninfected clams, both originating from the same lake, did not differ in their mean PBD. This indicated that burrowing of the experimental clams affected parasitism rather than the parasites altering burrowing of the clams. In line with the experimental result, we observed a significant positive correlation between PBD and the abundance of P. rylovi also among clams collected from 2 natural A. piscinalis populations.

Mature worms of Stephanoprora amurensis sp. nov. were obtained in an experimental study of its life cycle. In the Russian southern Far East, this trematode circulates using freshwater snails Parajuga subtegulata, freshwater fish and birds as the first, second intermediate and final hosts, respectively. Stephanoprora amurensis sp. nov. differs from the well-known representatives of Stephanoprora in a number of morphometric indicators of the developmental stages. The validity of the species was also confirmed by nuclear and mitochondrial DNA markers. In addition, new genetic data were obtained for Echinochasmus suifunensis and Echinochasmus milvi. An analysis of phylogenetic relationships within Echinochasmidae based on the 28S rRNA gene and ITS2 region identified two clusters, one of which combines species of Echinochasmus with 20–22 collar spines and short-tailed cercariae, and the other which includes Stephanoprora spp. and a number of representatives of Echinochasmus with 24 collar spines and long-tailed cercariae. The results of phylogenetic analysis based on ITS2 data show interfamily level of differences between the two clusters and intergeneric differentiation between the three subclusters uniting the species of Stephanoprora and Echinochasmus.

EhNCABP166 is an Entamoeba histolytica actin-binding protein that localizes to the nucleus and cytoplasm. Bioinformatic analysis of the EhNCABP166 amino acid sequence shows the presence of 3 bipartite nuclear localization signals (NLS) and a nuclear export signal (NES). The present study aimed to investigate the functionality of these signals in 3 ways. First, we fused each potential NLS to a cytoplasmic domain of ehFLN to determine whether the localization of this domain could be altered by the presence of the NLSs. Furthermore, the localization of each domain of EhNCABP166 was determined. Similarly, we generated mutations in the first block of bipartite signals from the domains that contained these signals. Additionally, we added an NES to 2 constructs that were then evaluated. We confirmed the intranuclear localization of EhNCABP166 using transmission electron microscopy. Fusion of each NLS resulted in shuttling of the cytoplasmic domain to the nucleus. With the exception of 2 domains, all of the evaluated domains localized within the nucleus. A mutation in the first block of bipartite signals affected the localization of the domains containing an NLS. The addition of an NES shifted the localization of these domains to the cytoplasm. The results presented here establish EhNCABP166 as a protein containing functional nuclear localization signals and a nuclear export signal.

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa, and toxoplasmosis is an important disease of both humans and economically important animals. With a limited array of drugs available there is a need to identify new therapeutic compounds. Aureobasidin A (AbA) is an antifungal that targets the essential inositol phosphorylceramide (IPC, sphingolipid) synthase in pathogenic fungi. This natural cyclic depsipeptide also inhibits Toxoplasma proliforation, with the protozoan IPC synthase orthologue proposed as the target. The data presented here show that neither AbA nor an analogue (Compound 20), target the protozoan IPC synthase orthologue or total parasite sphingolipid synthesis. However, further analyses confirm that AbA exhibits significant activity against the proliferative tachyzoite form of Toxoplasma, and Compound 20, whilst effective, has reduced efficacy. This difference was more evident on analyses of the direct effect of these compounds against isolated Toxoplasma, indicating that AbA is rapidly microbicidal. Importantly, the possibility of targeting the encysted, bradyzoite, form of the parasite with AbA and Compound 20 was demonstrated, indicating that this class of compounds may provide the basis for the first effective treatment for chronic toxoplasmosis.

Due to an increasing demand for natural products to control coccidiosis in broilers, we investigated the effects of supplementing a combination of ethanolic extracts of Artemisia annua and Curcuma longa in drinking water. Three different dosages of this herbal mixture were compared with a negative control (uninfected), a positive control (infected and untreated), chemical coccidiostats (nicarbazin+narazin and, later, salinomycin), vaccination, and a product based on oregano. Differences in performance (weight gain, feed intake, and feed conversion rate), mortality, gross intestinal lesions and oocyst excretion were investigated. Broilers given chemical coccidiostats performed better than all other groups. Broilers given the two highest dosages of the herbal mixture had intermediate lesion scores caused by Eimeria acervulina, which was higher than in broilers given coccidiostats, but less than in broilers given vaccination, oregano and in negative controls. There was a trend for lower mortality (P = 0·08) in the later stage of the growing period (23–43 days) in broilers given the highest dosage of herbal mixture compared with broilers given chemical coccidiostats. In conclusion, the delivery strategy of the herbal extracts is easy to implement at farm level, but further studies on dose levels and modes of action are needed.

Changes in population allele frequencies may be driven by several forces, including selection and drift, and are revealed only by sampling over many generations. Such studies, however, are rare for protist parasites. Microsatellite allele frequencies for 4 loci were followed in a population of Plasmodium mexicanum, a malaria parasite of lizards in California USA at 1 site from 1978 to 2010. Rapid turnover of the lizards indicates the parasite was studied for a minimum of 33 transmission cycles and possibly twice that number. Sample sizes ranged from 841 to 956 scored parasite clones per locus. DNA was extracted from frozen dried blood and blood removed from stained blood smears from the earliest years, and a verification study demonstrated DNA from the blood smears provided valid genetic data. Parasite prevalence and effective population size (Ne) dropped after 2000, remaining lower for the next decade. For 2 loci, allele frequencies appeared stable for the first 2 decades of the study, but changed more rapidly after the decline in prevalence. Allele frequencies changed more gradually for the other 2 loci. Genetic drift could account for changes in allele frequencies, especially after the drop in prevalence and Ne, but the force of selection could also have driven the observed patterns.

Although there is a plethora of cancer associated-factors that can ultimately culminate in death (cachexia, organ impairment, metastases, opportunistic infections, etc.), the focal element of every terminal malignancy is the failure of our natural defences to control unlimited cell proliferation. The reasons why our defences apparently lack efficiency is a complex question, potentially indicating that, under Darwinian terms, solutions other than preventing cancer progression are also important contributors. In analogy with host-parasite systems, we propose to call this latter option ‘tolerance’ to cancer. Here, we argue that the ubiquity of oncogenic processes among metazoans is at least partially attributable to both the limitations of resistance mechanisms and to the evolution of tolerance to cancer. Deciphering the ecological contexts of alternative responses to the cancer burden is not a semantic question, but rather a focal point in understanding the evolutionary ecology of host-tumour relationships, the evolution of our defences, as well as why and when certain cancers are likely to be detrimental for survival.

Among the variety of virulence factors of Entamoeba histolytica, an adherence lectin (Gal/GalNAc, 260 kDa) is known to mediate colonization and subsequent host responses. Gal/GalNAc lectin is universally recognized by the immune sera of patients with amoebic liver abscess. It plays a crucial role in cytolysis and phagocytosis of human and rat colonic mucin glycoproteins. The objective of the present study was to elucidate the role of antioxidants in E. histolytica Gal/GalNAc lectin-induced signals in the target epithelial cells. We have attempted to define a pathway in target cells, Henle-407 cells (human intestinal epithelial cell line), that could link this immunodominant antigen to a known biological pathway for target cell activation and triggering of subsequent disease pathology/parasite survival. Since several workers have demonstrated that cAMP and cGMP may act as important cellular signals for altering ion transport, so in the present study, cAMP and cGMP levels were measured in Henle-407 cells which showed significant increase at 15 min after stimulation. Elevated cAMP and cGMP levels are implicated in altered electrolyte transport and conductance. Results showed that there were increased levels of ROS and RNI which led to reduced activities of antioxidant enzymes – catalase, superoxide dismutase and glutathione peroxidase. Despite the increased glutathione (reduced) levels, the enzymes were not able to combat the damage caused by ROS and RNI. Thus, there was an increased local concentration of the free radicals and reduced activities of all the three enzymes which could damage the target cell in terms of cytoskeleton and permeability changes.

1. Observations indicate that the female uses the third pair of legs during deposition as an aid in forcing the larva through the vaginal orifice.

2. The young imago is able to free itself from the pupal case by dragging the case across a rough surface with the forelegs.

3. The act of copulation has been observed and described, including mounting, position and duration.

4. Evidence is presented to show that the insects are positively thermotropic and thigmotropic; negatively phototropic and hydrotropic.

5. The flies react positively to the odours of oil of cloves, camphor and ox gall; negatively to oil of wintergreen and amyl alcohol, and are indifferent to blood, fresh pigeon skin, pigeon scales, feathers, ammonia, oil of thyme, oil origanum, xylol and bergamot.

6. The length of life without food is found to be in direct proportion to the energy expended in motion, ranging from 77 to 109 hours.

7. The flies refuse to take unwarmed food.

8. The insect will not penetrate a perfectly smooth membrane, or one that is rancid.

9. According to data presented. Pseudolynchia maura will not live longer than 117 hours when artificially fed.

Alteration of the free amino acid pool of Hymenolepis diminuta was observed following incubation of worms with a single exogenous amino acid in vitro, recovery from which depended upon the initial magnitude of alteration. The free amino acid pool of both rat small intestine and H. diminuta was rendered imbalanced after feeding a single dietary dose of proline. Recovery was more rapid in the host than in the parasite tissues. The quantitative dynamics of amino acid change with time were examined. The effects of amino acid variation upon the uptake and incorporation of lysine-C14 into worm protein were examined in vitro. Four amino acids altered lysine incorporation; aspartate stimulated incorporation while ornithine, arginine and histidine reduced variously the absolute amount of lysine uptake but produced relative stimulation of lysine incorporation. These data indicate that, in the system examined, short term variation of the free amino acid pool is unlikely to affect protein synthesis in H. diminuta. Data were obtained suggesting the possible occurrence of certain amino acid inter conversions in H. diminuta.

This paper reports a longitudinal study, conducted in 1989/90, of 1893 children aged 5 to 14 years in 9 poor urban areas of the city of Salvador (population 2·44 million), capital of Bahia State in northeast Brazil. Stool examinations were performed to measure nematode infection and reinfection 9 months after treatment, and an extensive questionnaire was applied to collect information on each child and on the conditions of the household. Comparison of areas with different levels of infrastructure showed the following trends as the level of community sanitation improved: clustering of cases by household became more significant, predisposition of individuals to reinfection and to heavy infection became more marked, and infections with different species were increasingly aggregated in the same individuals. These results suggest that sewerage and drainage can significantly reduce transmission of intestinal nematode infections in the public domain, but that other measures are required to control transmission within the household.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50 % loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils. Treatment of these animals with CGP 20376, with autopsy 2 weeks later, resulted in a small macrofilaricidal effect (28% worm death). Some reduction in the motility of living worms recovered from CGP 20376-treated gerbils was also evident. Good survival of control worms recovered 2 weeks post-implantation suggests that male O. volvulus might survive longer subcutaneously. The prospects for undertaking meaningful drug evaluations against O. volvulus in vitro (and in vivo in rodents) is therefore promising, but because of parasite availability will probably be limited to those compounds which have shown potential in other filarial test systems.

The intrategumental pH in adult male Schistosoma mansoni as measured with pH-sensitive microelectrodes is between 7.0 and 7.2, a value about one pH unit more alkaline than expected for equilibrium. This value is maintained for at least 4 h after isolation in media buffered with Hepes or CO2/ with or without serum present. CCCP (1 μM) and FCCP (10 μM) cause rapid acidification. DNP (1 mM) and Na-orthovanadate (1 mM) acidify but also cause significant depolarization of the tegument. NH (20 mM) depolarizes and acidifies the tegument with no evidence of transient alkalinization. High K+ (25 mM) accelerates the acidification. Removal of the NH+4 causes a large transient further acidification with recovery requiring as long as 40 min. High K+ (25 mM) delays the early stage of the recovery. Presence of CO2/HCO in the incubation medium does not accelerate the recovery rate nor does SITS (100 μM) inhibit the recovery. Intrategumental Na+ is elevated after an acid load and amiloride (3 mM) as well as low Na+ medium interfere with recovery from the acid load indicating that a Na+–H+ exchanger may be present in the tegumental membrane.

We have established an amplified fragment length polymorphism (AFLP) protocol for identifying anonymous polymorphic loci of the malarial parasite, Plasmodium falciparum. The method consists of the following steps (i) digestion and ligation in one reaction; (ii) selective fluorescence forward primers labelled; (iii) PCR products resolved in polyacrylamide gels using the ABIPRISMTM 377 XL DNA sequencer and, (iv) the use of GenescanTM software to size the fragments. This standardized protocol distinguished between 2 standard reference clones of P. falciparum from West African and Southeast Asian and 2 Central African isolates from patients with clinical malaria. The AFLP protocol resulted in evenly distributed and reproducible band patterns for amplified fragments ranking from 163 to 489 bp long ±0·5 S.D. The primer Tru ACA labelled with the phosphoramidite 6-carboxifluorescein (FAM-blue) was easy to interpret, with a maximum of 53 bands per clone and of 81 per isolate (mixed falciparum populations) whereas the primer Tru AG labelled with the hexachlorinated analogue (HEX-green) showed a less clear pattern of bands and reproducibility than Tru ACA.