Helminth parasites of the wood mouse Apodemus sylvaticus in Southern England: levels of infection, species richness and interactions between species.

Helminth parasites of the wood mouse, Apodemus sylvaticus (n = 440), were surveyed in five localities, comprising woodland and grassland sites, in Southern England. Seven species of helminths were identified, among which Heligmosomoides polygyrus and Syphacia stroma were dominant (prevalence = 79.1% and 54.1%, respectively). Less common species were the trematode Corrigia vitta (14.8%), cestodes Catenotaenia pusilla (8.4%), Hydatigera taeniaeformis (4.1%) and Microsomacanthus crenata (3.4%) and the nematode Aonchotheca murissylvatici (0.2%). Differences in prevalences between localities were found for H. polygyrus, H. taeniaeformis and M. crenata and in abundances of H. polygyrus, S. stroma and C. vitta. Age-dependent increases in both parameters were identified among species and for helminth species richness. The only species to show significant host sex bias was S. stroma with prevalence values being higher in male mice. A number of different methods for exploiting raw data, and data corrected for significant confounding factors, were used to determine whether there were significant associations (prevalence) between species or quantitative interactions (abundance). The strongest evidence for a positive association was shown in concurrent infections with the trematode C. vitta and the cestode C. pusilla (significant in the whole dataset and evident in each locality, both sexes and both age classes). The abundance of C. pusilla was also higher in mice with C. vitta and vice versa. Overall, however, there was little support for associations or quantitative interactions between species, especially after data had been corrected for significant extrinsic/intrinsic factors, and we conclude that the helminths of wood mice in these communities are largely non-interactive and hence, perhaps better referred to as assemblages.

The effect of conventional preservatives on spicule length of Heligmosomoides bakeri (Nematoda, Heligmosomidae).

Nematode spicules vary in shape and size even between closely related species and, therefore, constitute key characters in nematode taxonomy for distinguishing between species. Spicules are seldom measured on fresh specimens, but rather at some time after extraction from culled hosts and after a period of preservation of the worms in chemical fixatives or by freezing. We carried out two experiments to assess the effects of freezing in Hanks' balanced salt solution, 70% or 80% ethanol and 10% formalin (both of the latter at room temperature and after storage at -80°C) on spicule length of Heligmosomoides bakeri at two time intervals after extraction from mice (Experiment 1, one and four weeks; Experiment 2, one and four months). In Experiment 1, no significant differences were detected, although there was some variation between treatments and over time. In Experiment 2, spicule length varied significantly between treatments and over time, the greatest shrinkage being in 80% ethanol and the least in 10% formalin. However, overall variation in spicule length was very limited, accounting for no more than 5.03% change in length over time and 4.95% between treatments at any of the periods of assessment. Therefore, while whole nematodes can shrivel and shrink in preservatives, making many measurements unreliable, our data indicated that spicule lengths are very little changed by preservation techniques over time, and so spicule length remains as a reliable taxonomic character.

Spatial interactions between two nematode species along the intestine of the wood mouse Apodemus sylvaticus from woodland and grassland sites in southern England.

The distributions of the nematode parasites Heligmosomoides polygyrus and Syphacia stroma were quantified in three equal-length sections along the intestine of wood mice (Apodemus sylvaticus) trapped in three different locations in the south of England. The distribution of H. polygyrus did not change in the presence of S. stroma, this species being largely confined to the anterior third of the intestine, whether S. stroma was or was not present. However, while in single infections with S. stroma, worms were equally distributed in the anterior and middle sections of the intestine, in the presence of H. polygyrus, a higher percentage of worms was located in the middle section. This was a dose-dependent response by S. stroma to increasing worm burdens with H. polygyrus, and even relatively low intensities of infection with H. polygyrus (e.g. ≤10 worms) were sufficient to cause a posterior redistribution of S. stroma into the middle section. A similar posterior shift in the percentage distribution of S. stroma in the intestine was evident in juvenile and mature mice of both sexes, and in mice from all three study sites. The ecological significance of these results is discussed.

Developing novel anthelmintics: the stability of cysteine proteinase activity in a supernatant extract of papaya latex.

Plant derived cysteine proteinases (CPs) have long been known to possess anthelmintic properties but have attracted renewed attention recently because of the acute need to discover novel methods for controlling helminth infections as a result of increasing drug resistance. However, surprisingly little is known about the stability of these proteins under typical storage and in vivo exposure conditions. We found that CPs in a supernatant preparation from papaya latex (PLS) were stable during the initial refinement process and when stored under low temperatures, but lost activity during dialysis and within 7 days of storage when kept at ambient temperature (18-20 °C). The enzyme activity in PLS was not affected by repeated freeze-thaw cycles and was also stable under typical in vitro assay conditions at 37 °C used for quantifying effects on helminths. Active enzyme activity was still detectable in the colon 3-4 h after oral administration in rodent models.

The development of spicules in Heligmosomoides bakeri (Nematoda, Heligmosomidae).

The spicules of male parasitic nematodes are key morphological features, which vary between species in shape and length and are used often for species identification. However, little is known about spicules and particularly if/how their length varies during growth. We first assessed the degree of variation in spicule length of male Heligmosomoides bakeri 21 days post infection (PI), and then in two follow-up experiments measured spicule lengths at half daily/daily intervals between days 6 and 14 PI. Mean spicule length in 21-day worms was 0.518 mm with a range of 94 µm, and variation between the two spicules of individual worms from 2 to 32 µm. Spicules were first detectable on day 6-6.5, after which their lengths increased until day 7 PI (mean = 0.61 and 0.59). This was followed by significant contraction, initially relatively quickly over the following 48 h and then more slowly over a longer period, stabilizing by days 10-14, with only minor further reduction in length. We conclude that the length of spicules varies significantly over the first few days after they have formed, and, consequently, the age of worms is an important factor for consideration when spicule lengths are measured for experimental/diagnostic or taxonomical purposes.

Entamoeba infections and associated risk factors among migrant workers in Peninsular Malaysia.

The influx of low skilled migrant workers to Malaysia from low socio-economic countries where gastrointestinal parasitic infections are prevalent has raised concerns about transmission to the local population. Three methods for detection (serology, microscopy and molecular techniques) were utilized to identify Entamoeba infections amongst the targeted cohort and determine risk factors associated with infection. Serological screening of 484 migrant workers from five working sectors in Peninsular Malaysia using IgG4 ELISA based on the rPPDK antigen showed an overall seroprevalence of 7.4% (n = 36; CL95 = 5.3-10.1%) with only one factor statistically associated with seropositivity of anti-amoebic antibodies, i.e. years of residence in Malaysia (χ2 1 = 4.007, p = 0.045). Microscopic examination of 388 faecal samples for protozoan cysts and trophozoites showed a slightly higher prevalence (11.6%; n=45; CL95: 8.4-14.8%). Meanwhile, amplification of the 16S rDNA gene detected two species i.e. Entamoeba dispar (23/388; 5.9%; CL95: 3.6-8.3%) and E. histolytica (11/388; 2.8%; CL95: 1.2-4.5%) and mixed infections with both parasites in only three samples (3/388; 0.8%; CL95: 0.2-2.2%). Entamoeba dispar infection was significantly associated with those employed in food and domestic services (χ2 4 = 12.879, p = 0.012). However, none of the factors affected the prevalence of E. histolytica infection. Despite the low prevalence of E. histolytica in faecal samples of the study cohort, the presence of this pathogenic parasite still poses potential public health risks and calls for tighter control strategies based on better availability of chemotherapeutic treatment and accessibility to appropriate health education.

A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro.

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional 'ethnoveterinary' medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12-0.22µM. At concentrations above 6.25µM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl-l-leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35-106.31µM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

The anthelmintic efficacy of natural plant cysteine proteinases against the equine tapeworm, Anoplocephala perfoliata in vitro.

Papaya latex has been demonstrated to be an efficacious anthelmintic against murine, porcine, ovine and canine nematode parasites, and even those infecting poultry, and it has some efficacy against rodent cestodes. The active ingredients of papaya latex are known to be cysteine proteinases (CPs). The experiments described in this paper indicate that CPs in papaya latex, and also those in pineapples, are highly efficacious against the equine cestode Anoplocephala perfoliata in vitro, by causing a significant reduction in motility leading to death of the worms. The susceptibility of A. perfoliata to damage by CPs was considerably greater than that of the rodent cestodes Hymenolepis diminuta and H. microstoma. Our results are the first to report anthelmintic efficacy of CPs against an economically important equine helminth. Moreover, they provide further evidence that the spectrum of activity of CPs is not restricted to nematodes and support the idea that these plant-derived enzymes can be developed into useful broad-spectrum anthelmintics.

The anthelmintic efficacy of natural plant cysteine proteinases against the rat tapeworm Hymenolepis diminuta in vivo.

Hymenolepis diminuta is a natural parasite of the common brown rat Rattus norvegicus, and provides a convenient model system for the assessment of the anthelmintic activity of novel drugs against cestodes. The experiments described in this paper indicate that treatment of rats infected with H. diminuta with a supernatant extract of papaya latex, containing a mixture of four cysteine proteinases, was moderately efficacious, resulting in a significant, but relatively small, reduction in worm burden and biomass. However, faecal egg output was not affected by treatment. In our experiments these effects were only partially dose-dependent, although specific inhibition by E-64 confirmed the role of cysteine proteinases as the active principles in papaya latex affecting worm growth but not statistically reducing worm burden. Data collected for a further 7 days after treatment indicated that the effects of papaya latex supernatant on worm loss and on worm growth were not enhanced. Our findings provide a starting point for further refinement in formulation and delivery, or assessment of alternative natural plant-derived cysteine proteinases in efforts to develop these naturally occurring enzymes into broad-spectrum anthelmintics, with efficacy against cestodes as well as nematodes.

Bank voles (Myodes glareolus) and house mice (Mus musculus musculus; M. m. domesticus) in Europe are each parasitized by their own distinct species of Aspiculuris (Nematoda, Oxyurida).

The molecular phylogeny and morphology of the oxyuroid nematode genus Aspiculuris from voles and house mice has been examined. Worms collected from Myodes glareolus in Poland, Eire and the UK are identified as Aspiculuris tianjinensis, previously known only from China, while worms from Mus musculus from a range of locations in Europe and from laboratory mice, all conformed to the description of Aspiculuris tetraptera. Worms from voles and house mice are not closely related and are not derived from each other, with A. tianjinensis being most closely related to Aspiculuris dinniki from snow voles and to an isolate from Microtus longicaudus in the Nearctic. Both A. tianjinensis and A. tetraptera appear to represent recent radiations within their host groups; in voles, this radiation cannot be more than 2 million years old, while in commensal house mice it is likely to be less than 10,000 years old. The potential of Aspiculuris spp. as markers of host evolution is highlighted.

The Status of Heligmosomoides americanus, Representative of an American Clade of Vole-Infecting Nematodes.

Heligmosomoides americanus is shown by molecular phylogenetic analysis of 3 nuclear (28S, ITS1, and ITS2) and 2 mitochondrial (cytochrome oxidase 1 and cytochrome b) loci to be a distinct species of heligmosomid nematode with a long-independent evolutionary history, and not a subspecies of Heligmosomoides polygyrus . Rather than being a recent arrival in North America, the species probably originated as a Beringian immigrant with the host vole Phenacomys, approximately 2 million years ago (MYA).

The anthelmintic efficacy of natural plant cysteine proteinases against Hymenolepis microstoma in vivo.

Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections in vivo. Hymenolepis microstoma is a natural parasite of house mice, and provides a convenient model system for the assessment of novel drugs for anthelmintic activity against cestodes. The experiments described in this paper indicate that treatment of H. microstoma infections in mice with the supernatant of papaya latex (PLS), containing active cysteine proteinases, is only minimally efficacious. The statistically significant effects seen on worm burden and biomass showed little evidence of dose dependency, were temporary and the role of cysteine proteinases as the active principles in PLS was not confirmed by specific inhibition with E-64. Worm fecundity was not affected by treatment at the doses used. We conclude also that this in vivo host-parasite system is not sensitive enough to be used reliably for the detection of cestocidal activity of compounds being screened as potential, novel anthelmintics.

The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes.

We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections.

Behavioural changes in the flour beetle Tribolium confusum infected with the spirurid nematode Protospirura muricola.

We examined changes to the behaviour of flour beetles, Tribolium confusum, infected with the rodent stomach worm, the spirurid Protospirura muricola, in the context of the 'Behavioural Manipulation Hypothesis'. Trobolium confusum infected with the third-stage infective larvae of P. muricola showed consistently altered patterns of behaviour. Relative to uninfected beetles, over a measured time period, beetles infected with P. muricola were likely to move over a shorter distance, when moving their speed of movement was slower, they were more likely to stay in the illuminated area of their environment, more likely to emerge from darkened areas into the illuminated areas, and their longevity was significantly shortened. The changes in behaviour, as reflected in effects on speed of movement, were only evident among beetles that actually harboured infective cysts and not among those carrying younger infections when the larvae within their haemocoels would have been at an earlier stage of development and not yet capable of infecting the definitive murine hosts. We discuss whether these changes would have made the beetles more susceptible to predation by rodents, and specifically by the omnivorous eastern spiny mouse, Acomys dimidiatus, the natural definitive host of this parasite in Egypt, from where the P. muricola isolate originated, and whether they support the Behavioural Manipulation Hypothesis or reflect parasite-induced pathology.

The anthelmintic efficacy of natural plant cysteine proteinases against two rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma in vitro.

Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74 µM, respectively, and for pre-adult worms=199 and 260 µM, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772 µM, respectively, and for pre-adult worms=1374 and 1332 µM, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected.

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

Understanding the role of antibodies in murine infections with Heligmosomoides (polygyrus) bakeri: 35 years ago, now and 35 years ahead.

The rodent intestinal nematode H.p.bakeri has played an important role in the exploration of the host-parasite relationship of chronic nematode infections for over six decades, since the parasite was first isolated in the 1950s by Ehrenford. It soon became a popular laboratory model providing a tractable experimental system that is easy to maintain in the laboratory and far more cost-effective than other laboratory nematode-rodent model systems. Immunity to this parasite is complex, dependent on antibodies, but confounded by the parasite's potent immunosuppressive secretions that facilitate chronic survival in murine hosts. In this review, we remind readers of the state of knowledge in the 1970s, when the first volume of Parasite Immunology was published, focusing on the role of antibodies in protective immunity. We show how our understanding of the host-parasite relationship then developed over the following 35 years to date, we propose testable hypotheses for future researchers to tackle, and we speculate on how the new technologies will be applied to enable an increasingly refined understanding of the role of antibodies in host-protective immunity, and its evasion, to be achieved in the longer term.

The mucosal response of hamsters exposed to weekly repeated infections with the hookworm Ancylostoma ceylanicum.

An experiment was carried out to assess mucosal changes in hamsters exposed to weekly repeated low-intensity infections with the hookworm Ancylostoma ceylanicum. The experiment included control groups of naïve, uninfected hamsters and groups that received a single-pulse primary infection. Changes in the intestinal architecture and in the density of inflammatory cells in the mucosa, including mast cells, goblet cells, Paneth cells and eosinophils were examined in relation to changes in hookworm burdens. As in the single-pulse primary infection, hamsters exposed to repeated infections responded with marked changes in the intestinal architecture and in mucosal populations of inflammatory cells. However, there were distinct differences in the kinetics of the responses to these two types of infection (primary single-pulse and repeated). The reduction in villous height and the increase in crypt depth in animals exposed to repeated infections were both initially slower but eventually equalled and exceeded the responses in hamsters given a chronic primary infection, despite the presence of fewer adult worms in the former. Similarly, changes in the mitotic figures of epithelial cells in the mucosa and the mast cell response were both initially slower and less intense in repeatedly infected hamsters, but eventually exceeded the response to primary infection. Furthermore, the eosinophil response was found to be initially greater in repeated infections and overall more persistent. In contrast, both goblet and Paneth cell responses were less marked in repeatedly infected animals compared to those carrying a primary infection. These results are discussed in the context of host protective resistance to infection with A. ceylanicum.

The anthelmintic efficacy of papaya latex in a rodent-nematode model is not dependent on fasting before treatment.

In earlier studies of the anthelmintic activity of plant cysteine proteinases (CPs), a period of food deprivation was routinely employed before administration of CPs, but there has been no systematic evaluation as to whether this does actually benefit the anthelmintic efficacy. Therefore, we assessed the effect of fasting on the efficacy of CPs from papaya latex (PL) against Heligmosomoides bakeri in C3H mice. We used a refined, supernatant extract of papaya latex (PLS) with known active enzyme content. The animals were divided into three groups (fasted prior to treatment with PLS, not fasted but treated with PLS and fasted but given only water). The study demonstrated clearly that although food deprivation had been routinely employed in much of the earlier work on CPs in mice infected with nematodes, fasting has no beneficial effect on the efficacy of PLS against H. bakeri infections. Administration of CPs to fed animals will also reduce the stress associated with fasting.