Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode, Protospirura muricola, in vitro and in vivo

Cysteine proteinases from the fruit and latex of plants, including papaya, pineapple and fig, were previously shown to have a rapid detrimental effect, in vitro, against the rodent gastrointestinal nematodes, H eligmosomoides polygyrus (which is found in the anterior small intestine) and Trichuris miti,is (which resides in the caecum). Proteinases in the crude latex of papaya also showed anthelmintic efficacy against both nematodes in vivo. In this paper, we describe the in vitro and in vivo effects of these plant extracts against the rodent nematode, Protospirua muricola, which is found in the stomach. As in earlier work, all the plant cysteine proteinases examined, with the exception of actinidain from the juice of kiwi fruit, caused rapid loss of motility and digestion of the cuticle, leading to death of the nematode in vitro. In vivo, in contrast to the efficacy against H. polygyrus and T. muris, papaya latex only showed efficacy against P. muricola adult female worms when the stomach acidity had been neutralized prior to administration of papaya latex. Therefore, collectively, our studies have demonstrated that, with the appropriate formulation, plant cysteine proteinases have efficacy against nematodes residing throughout the rodent gastrointestinal tract

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock

The mucosal cellular response to infection with Ancylostoma ceylanicum

Although hookworms are known to stimulate inflammatory responses in the intestinal mucosa of their hosts, there is little quantitative data on this aspect of infection. Here we report the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum. Infection resulted in a marked increase in goblet cells in the intestinal mucosa, which was dependent on the number of adult worms present and was sustained as long as worms persisted (over 63 days) but returned to baseline levels within 7 days of the removal of worms by treatment with ivermectin. Increased mast cell responses were also recorded. Levels were again dependent on the intensity of worm burdens and lasted as long as 63 days after infection. When worms were eliminated, mast cell numbers took over 2 weeks to return to normal. Paneth cell numbers fell soon after infection, the degree of reduction being dependent on the worm burden. After clearance of worms, Paneth cell numbers returned to normal within a week, but then rebounded and numbers rose to higher levels than those in control naıve animals. The time course of the response was similar whether animals experienced a chronic low-intensity infection without loss of worms or a higher intensity infection during the course of which worm burdens were gradually reduced. Clearly, A. ceylanicum was able to induce a marked inflammatory response in its host’s intestine which was sustained for over 9 weeks after infection, and which hamsters appeared able to tolerate well. Our data draw attention to the resilience of hookworms which, unlike many other nematodes, are able to survive for many weeks in a highly inflamed intestinal tract

Density-dependent effects on the survival and growth of the rodent stomach worm Protospirura muricola in laboratory mice

The spirurid nematode, Protospirura muricola, is of intrinsic interest as a rodent model of gastric nematode infections. Since worm burdens can be very heavy in nature, density dependent processes may constrain parasite growth. Laboratory mice (BKW) were exposed to varying doses of infective larvae of P. muricola in the range 5 to 40 third-stage larvae (L3), in four separate experiments in which progressively higher doses were utilized. All mice were culled 60 days after infection and a total of 518 worms (226 male and 292 female worms) was recovered, measured and weighed. Overall survival was 58.9%, but survival declined significantly with increasing dose by approximately 21% (from 66% at 5 L3 per mouse to 52% at 40 L3 per mouse). The length and weight of worms correlated positively in both sexes. Total worm biomass increased linearly with increasing numbers of worms. However, whilst the length and weight of male worms declined with increasing worm burden (8.4 and 24.6% respectively), female worms were less affected, only length showing a significant reduction with increasing parasite burden (16.0%). Therefore, increasing worm burdens impeded growth of P. muricola, but reduction in length and weight were relatively small in relation to the overall size of this nematode. Increasing worm burdens were associated with loss of host weight and reduction in stomach weight and worm burdens in excess of 20 exerted a measurable cost to the host, which in the field, may be associated with loss of overall host fitness

The effect of the hookworm Ancylostoma ceylanicum on the mucosal architecture of the small intestine in hamsters

Hookworms are known to cause marked changes to the intestinal mucosa, especially in relation to erosion of the villi. However, since the development of enteropathy has not been examined thoroughly through quantitative experiments on infected animals, the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum are reported. Changes to intestinal architecture were first apparent between 12 and 14 days after infection, and then increased in intensity for 3–4 weeks, persisting for as long as worms were present (>63 days). Following infection, the height of villi declined from a mean of 1002 μm in naïve controls to less than 200 μm and as low as 18 μm in one case. The depth of the crypts of Lieberkuhn increased from a baseline value of 166 μm in naïve controls to in excess of 600 μm within 6 weeks of infection. Mitotic figures had a baseline value of 5.5 per villus-crypt unit, and this rose to in excess of 25 in some experiments. Changes were dependent on the intensity of the parasite burden on day 20, but by 30 days after infection changes in all three values were maximal and density-dependent relationships were no longer clearly apparent. Villus height and crypt depth returned to near normal values within a week of the removal of worms, although group means for both remained different from naïve controls for at least 3 weeks after treatment. Cellular division, as reflected in numbers of mitotic figures, stayed elevated for over 5 weeks after removal of worms. The results suggest that enteropathy in hookworm infections stems from a combination of intestinal immune responses and from the grazing activities of the adult worms on the mucosal surface, but is not sufficient per se for expulsion of this parasit

Cysteine proteinases from papaya (Carica papaya) in the treatment of experimental Trichuris suis infection in pigs: two randomized controlled trials

Background: Cysteine proteinases (CPs) from papaya (Carica papaya) possess anthelmintic properties against human soil-transmitted helminths (STH, Ascaris lumbricoides, Trichuris trichiura and hookworm), but there is a lack of supportive and up-to-date efficacy data. We therefore conducted two randomized controlled trials in pigs to assess the efficacy of papaya CPs against experimental infections with T. suis. Methods: First, we assessed efficacy by means of egg (ERR) and adult worm reduction rate (WRR) of a single-oral dose of 450 μmol active CPs (CP450) against low (inoculum of 300 eggs) and high (inoculum of 3,000 eggs) intensity T. suis infections and compared the efficacy with those obtained after a single-oral dose of 400 mg albendazole (ALB). In the second trial, we determined and compared the efficacy of a series of CP doses (45 [CP45], 115 [CP115], 225 [CP225], and 450 [CP450] μmol) against high intensity infections. Results: CP450 was highly efficacious against both levels of infection intensity, resulting in ERR and WRR of more than 97%. For both levels of infection intensity, CP450 was significantly more efficacious compared to ALB by means of WRR (low infection intensity: 99.0% vs. 39.0%; high infection intensity; 97.4% vs. 23.2%). When the efficacy was assessed by ERR, a significant difference was only observed for high intensity infections, CP450 being more efficacious than ALB (98.9% vs. 59.0%). For low infection intensities, there was no significant difference in ERR between CP450 (98.3%) and ALB (64.4%). The efficacy of CPs increased as a function of increasing dose. When determined by ERR, the efficacy ranged from 2.1% for CP45 to 99.2% for CP450. For WRR the results varied from -14.0% to 99.0%, respectively. Pairwise comparison revealed a significant difference in ERR and WRR only between CP45 and CP450, the latter being more efficacious. Conclusions: A single dose of 450 μmol CPs provided greater efficacy against T. suis infections in pigs than a single-oral dose of 400 mg ALB. Although these results highlight the possibility of papaya CPs for controlling human STH, further development is needed in order to obtain and validate an oral formulation for human application

Helminths of the sand lizard, Lacerta agilis (Reptilia, Lacertidae), in the Palaearctic: faunal diversity and spatial patterns of variation in the composition and structure of component communities

We studied variation in the structure of component communities of helminths in sand lizards, Lacerta agilis, from 30 localities in the Ukraine and Bulgaria. Thirty-five separate samples of lizards, with a total of 661 completely censused infracommunities, yielded 30 helminth species (4 cestodes, 10 trematodes, 3 acanthocephalans and 13 nematodes). In its range within the Ukraine, L. agilis serves as the final host for 13 species of which only 3 (S. lacertae, S. hoffmanni and P. molini) can be considered as lizard specialists. A characteristic feature of these helminth component communities was thelarge proportion of heteroxeneous helminth species for which L. agilis serves as paratenic host. Sand lizards in the meadow steppeland zone were primarily parasitized by larval helminths that represented a major proportion of the total number of all worms recovered while those sampled in the grassland}forest transition zone were characterized by substantially higher proportions of adult helminths using lizards as final hosts. However, L. agilis was parasitized by a much higher proportion of lizard specialists in the `typical ' habitats of the meadow steppeland zone as opposed to those located in `marginal' habitats in the grassland/forest transition zone, where helminths were shared to a greater extent with amphibian hosts

Local variation in helminth burdens of Egyptian spiny mice (Acomys cahirinus dimidiatus) from ecologically similar sites: relationships with hormone concentrations and social behaviour

Populations of Egyptian spiny mice (Acomys cahirinus dimidiatus) in a fragmented montane wadi system in the Sinai showed significant differences in the abundance of gut helminths. Differences in parasite load between populations were positively associated with measures of androgen activity but showed no significant relationship with glucocorticoid activity. Social discrimination tests with adult males from different wadis showed that those from sites with greater helminth abundance were less likely to investigate odours from other males and were less aggressive when subsequently interacting with the odour donors. Subjects showed markedly more investigation towards the odours of males from distant wadis compared with those from their own or immediately neighbouring wadi, but were less aggressive when confronted with odour donors from distant wadis. Despite this, there was a positive relationship between the amount of investigation towards distant male odour and subsequent aggression towards the male. While aggressiveness was positively associated with measures of androgen and glucocorticoid activity, no significant relationship emerged with individual helminth infection. Thus aggressiveness appeared to relate to overall local population levels of infection rather than individual challenge