Transport of Schistosoma japonicum cercariae and the feasibility of niclosamide for cercariae control

Field experiments were conducted in southwestern China to estimate the distances over which cercariae of Schistosoma japonicum are transported and remain infective in village irrigation ditches and to determine the feasibility of interrupting this transport using niclosamide, the molluscicide commonly used for snail control in this environment. The number of worms recovered from bioassay mice downstream from experimentally introduced cercariae, but without niclosamide, decreased about 90% over the first 100 m of ditch and then remained essentially constant for the remaining 300 m of the ditch that was monitored. The niclosamide experiments resulted first in the finding that it was possible to achieve ditch average concentrations close to the target values of 0.25 ppm, but that concentrations at any particular location varied by a factor of up to 3. There was no correlation between water turbidity and niclosamide concentration. An average water concentration of 0.29 ppm resulted in no viable cercariae after a contact time of about 1 min and a travel distance of only 10 m. We regard these results as proof of the feasibility of chemical interruption of transport of S. japonicum cercariae although the toxicity of niclosamide to fish at the levels used here make it a questionable candidate for routine use.     

A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: The influence of the ages of the cercariae and of the host

A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: the influence of the ages of the cercariae and of the host. International Journal for Parasitology 3: 789–794. The number of cercariae of S. mansoni which die during penetration of mouse abdominal skin steadily increases with age following emergence from the snail. An initial mortality level of about 30 per cent is observed for 2-h-old cercariae which rises to 50 per cent at 8 h and 85 per cent at 24 h. These increased losses in the skin are shown to account substantially for the known decrease in infectivity which accompanies ageing of cercariae. The number of cercariae which die in the skin of very young mice (2 days old) is less than one-third of the level in adult mice. Losses in the skin increase with age of the host up to about 28–35 days. This increased mortality in the skin is shown to account for the observed age resistance of mice, where fewer cercariae mature into adult worms in mice of 1 month or more, than in very young mice.     

The molluscicidal activity of niclosamide (Bayluscide WP70(R)) on Melanoides tuberculata (Thiaridae), a snail associated with habitats of Biomphalaria glabrata (Planorbidae)

The aim of this study was to determine the toxicity of niclosamide (Bayluscide (R)) on Melanoides tuberculata and Biomphalaria glabrata under laboratory conditions. The latter species is the intermediate host of Schistosoma mansoni (Sambon 1917). M. tuberculata was successfully used as competitor of B. glabrata in biological control programs in French West Indies. Both molluscicide and biological control using M. tuberculata have proved to be successful in reducing the population density of B. glabrata. The associated use of molluscicide in this area would be an effective measure if M. tuberculata were less susceptibility to the molluscicide than B. glabrata. Three hundreds individuals each of B. glabrata and of M. tuberculata, collected in Sumidouro, State of Rio de Janeiro, were used in the experiment. The molluscs were exposed to 14 different concentrations of niclosamide as recommended by the World Health Organization. Probit analysis was used to determine the LC 50 and LC 90. The LC 50 and LC 90 values for B. glabrata were 0.077 mg/l and 0.175 mg/l, respectively and the LC 50 and LC 90 values for M. tuberculata were 0.082 mg/l and 0.221 mg/l respectively. As the lethal concentrations of niclosamide were approximately the same to both species, this could be a disadvantage when controlling B. glabrata with niclosamide in an area of M. tuberculata occurrence. It might therefore be preferable to utilize the latex extracted from the Euphorbia splendens, which presented a much higher efficiency for B. glabrata than to M. tuberculata.     

Cercarial tail loss in Echinostoma caproni: the influence of in vivo encystment and copper sulphate

Echinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l(-1) in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg(-1) H2O, Ca(2+) 20 mg l(-1)) at 23 degrees C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l(-1), 1000 mg l(-1) and 100 mg l(-1) CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59+/-5% of cercariae had lost their tails and only 4+/-1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72+/-3% of the cercariae began to form cysts within the snails.     

Vaccination with irradiated cercariae of Schistosoma mansoni preferentially induced the accumulation of interferon-gamma producing T cells in the skin and skin draining lymph nodes of mice

Cytokine response to schistosomula of Schistosoma mansoni was evaluated in the skin of mice during the initial 72 h following infection. These studies showed a significant increase in the levels of IL-4 and IL-10 message in the skin in areas of cercarial penetration. The IL-4 message was detectable in the skin as early as 8 h after infection and the message for IL-10 appeared from 16 h after infection. However, mRNA for IFN-gamma was undetectable in the skin samples for up to 72 h after infection with normal cercariae. In sharp contrast, vaccination with irradiated cercariae induced IFN-gamma and IL-2 responses in the skin within 24 h. Analysis of the cytokine profile of cells isolated from the skin during these early time points showed that T cells are probably not a source of IL-4 or IL-10 in the skin of mice infected with normal cercariae. However, in vaccinated animals, the majority of the IFN-gamma is derived from skin-residing T cells. In vaccinated animals, responses in the skin were mirrored in the skin-draining lymph nodes as well. Analysis of the CD4/CD8 ratio showed a significant decrease in the skin following vaccination suggesting an increase in CD8+ cells. Interestingly however, when vaccinated animals were challenged with normal cercariae, there was a significant reduction in IFN-gamma response in the skin and its draining lymph nodes. These results show that vaccination with irradiated cercariae of S. mansoni, preferentially induce the accumulation of IFN-gamma producing T cells in the skin and skin-draining lymph nodes of mice.     

日本血吸虫尾蚴钻穿不同宿主皮肤时新转变童虫的死亡情况

日本血吸虫尾蚴在钻穿7种哺乳动物宿主皮肤而转变为童虫时,均发生了自然死亡,而且童虫的死亡率依宿主种类或同一种类的不同品系而异。它们皮肤内平均童虫死亡率分别为:小鼠3.7—7.2％、大鼠6.3％、金黄仑鼠7.0％、长爪沙鼠5.1％、豚鼠6.9％、兔4.9％、恒河猴12.8％。实验证明日本血吸虫尾蚴亦能钻入不易感的鸟类——鸽的皮肤,虽有1/4童虫立即在其皮肤内发生死亡,但部分童虫可移行至肺,然后在肺中消亡。     

Effects of environmental calcium on fecundity and cercarial production of Biomphalaria glabrata (Say) infected with Schistosoma mansoni Sambon

Biomphalaria glabrata were reared in stock culture and subjected to either 7-day or 60-day acclimation periods in complex CaCO3 media with calcium values ranging from 1.5 mg/L to 75 mg/L. Following 60-day acclimation, snails from series I were each exposed to 8 miracidia of Schistosoma mansoni. Snails of series II were each exposed to a single miracidium. Snails of both experimental regimens were observed for mortality, growth, rate of infection, and number of cercariae shed. Series I snails were also monitored for fecundity during acclimation and following miracidial exposure. Calcium levels of 1.5 and 75 mg/L resulted in significant snail mortality. Shell growth and rates of infection were positively correlated with calcium maintenance level. Snails with high fecundity prior to miracidial exposure subsequently shed more cercariae. In contrast, post-exposure (PE) fecundity of snails reared in media with up to 30 mg/L Ca++ were negatively correlated with calcium level, infection rate, and number of cercariae shed. Maximal cercarial emergence occurred at 30 mg/L Ca++. These results suggest that environmental calcium affects both the distribution patterns of snail hosts of human schistosomes and the productivity of intramolluscan schistosome infection.     

Effects of copper sulphate on in vitro encystment of the cercariae of Echinostoma caproni

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.     

The chemical stimuli of human skin surface for the attachment response of Schistosoma mansoni cercariae

and 1986. The chemical stimuli of human skin surface for the attachment response of S mansoni cercariae. International Journal for Parasitology 16: 575–579. The stimuli for the S. mansoni attachment response were analysed by offering skin extracts and chemicals to cercariae through Millipore filters. The attachment stimulating components were contained in the hydrophilic skin extract. Whereas sugars had no stimulating activity, a mixture of electrolytes, lactate and urea had a weak, and amino acids a very strong, effect. The amino acids of the hydrophilic skin extract were qualitatively and quantitatively identified and their effectivity as pure chemicals compared with that of the skin extract. The acid amino acids and histidine and ornithine had a weak effect, whereas arginine alone stimulated as intensely as the whole hydrophilic skin extract. The specialization on arginine as the main attachment trigger could be interpreted as adaptation to the fact that creeping and penetrating cercariae secrete arginine with the contents of their post acetabular glands. Thus they might communicate their successful host identification to further cercariae.     

Delayed tail loss during the invasion of mouse skin by cercariae of Schistosoma japonicum

A traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm(2) of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.     

A comparative study of the death of schistosomula of Schistosoma haematobium and Schistosoma mansoni in the skin of mice and hamsters.

This study shows that some cercariae of S. haematobium and S. mansoni die during penetration of mouse or hamster skin. Approximately 30-38% of cercariae of both species die in mouse skin and 14-16% die in hamster skin. The greater number of cercariae which die in the skin of mice seems to account for the higher yield of adult worms recovered in hamsters. Adult worm recoveries from animals infected with S. haematobium were, however, only about half the worm recoveries from hosts infected with S. mansoni.     

Penetration of human skin by the cercariae of Schistosoma mansoni: an investigation of the effect of multiple cercarial applications

It has previously been postulated that L-arginine emitted by penetrating Schistosoma mansoni cercariae serves as an intraspecific signal guiding other cercariae to the penetration site. It was suggested that penetrating in groups offers a selective advantage. If this hypothesis is correct and group penetration at one site on the host offers an advantage, it would follow that at such a site, successive groups of cercariae would be able to penetrate skin in either greater numbers or at a faster rate. This prediction was tested by the use of an in vitro model of cercarial penetration based on the Franz cell and using human skin. It was demonstrated that there was no increase in the percentage of cercariae able to penetrate the skin with subsequent exposures. Consequently, it seems unlikely that the release of L-arginine by cercariae during penetration could have evolved as a specific orientation system based on a selective advantage offered by group penetration.     

Proteomic analysis of skin invasion by blood fluke larvae

Background

During invasion of human skin by schistosome blood fluke larvae (cercariae), a multicellular organism breaches the epidermis, basement membrane, and dermal barriers of skin. To better understand the pathobiology of this initial event in schistosome infection, a proteome analysis of human skin was carried out following invasion by cercariae of Schistosoma mansoni.

Methodology and Results

Human skin samples were exposed to cercariae for one-half hour to two hours. Controls were exposed to water used to collect cercariae in an identical manner, and punctured to simulate cercarial tunnels. Fluid from both control and experimental samples was analyzed by LC/MS/MS using a linear ion trap in “triple play” mode. The coexistence of proteins released by cercariae and host skin proteins from epidermis and basement membrane confirmed that cercarial tunnels in skin were sampled. Among the abundant proteins secreted by cercariae was the cercarial protease that has been implicated in degradation of host proteins, secreted proteins proposed to mediate immune invasion by larvae, and proteins implicated in protection of parasites against oxidative stress. Components of the schistosome surface tegument, previously identified with immune serum, were also released. Both lysis and apoptosis of epidermal cells took place during cercarial invasion of the epidermis. Components of lysed epidermal cells, including desmosome proteins which link cells in the stratum granulosum and stratum spinosum, were identified. While macrophage-derived proteins were present, no mast cell or lymphocyte cytokines were identified. There were, however, abundant immunoglobulins, complement factors, and serine protease inhibitors in skin. Control skin samples incubated with water for the same period as experimental samples ensured that invasion-related proteins and host protein fragments were not due to nonspecific degeneration of the skin samples.

Conclusions

This analysis identified secreted proteins from invasive larvae that are released during invasion of human skin. Analysis of specific host proteins in skin invaded by cercariae served to highlight both the histolytic events facilitating cercarial invasion, and the host defenses that attempt to arrest or retard invasion. Proteins abundant in psoriatic skin or UV and heat-stressed skin were not abundant in skin invaded by cercariae, suggesting that results did not reflect general stress in the surgically removed skin specimen. Abundant immunoglobulins, complement factors, and serine protease inhibitors in skin form a biochemical barrier that complements the structural barrier of the epidermis, basement membrane, and dermis. The fragmentation of some of these host proteins suggests that breaching of host defenses by cercariae includes specific degradation of immunoglobulins and complement, and either degradation of, or overwhelming the host protease inhibitor repertoire.     

Schistosoma mansoni: human skin ceramides are a chemical cue for host recognition of cercariae

Schistosoma mansoni cercariae recognize the human host with a sequence of behavioral responses particularly to chemical host cues. After attaching to the skin surface, cercariae are stimulated by so far unknown skin components to hold enduring contact with the skin and to start creeping towards entry sites. We studied the chemical stimulus of human skin for the cercarial enduring contact response by fractionation of human and pig skin surface extracts and offering the fractions to the cercariae via membrane filters. Enduring contact was stimulated exclusively by ceramides, specific lipids of the uppermost skin layers. This chemical cue differs from the 6 chemical host signals used by S. mansoni cercariae in other behavioral steps of host invasion, and thus underlines the specialization of S. mansoni cercariae particularly in chemical host signals. All together, the enduring contact response of the cercariae is, like the other phases of host invasion, well adapted to the chemical properties of human skin.     

Influence of saccharose on the development of cercariae from Schistosoma mansoni strains BH and SJ.

The development of cercariae from Schistosoma mansoni strains BH and SJ in Biomphalaria glabrata and Biomphalaria tenagophila treated with saccharose was studied. The molluscs were maintained in dechlorinated tap water containing 0.01% saccharose. After one week of treatment with saccharose, B. glabrata and B. tenagophila were exposed to ten S. mansoni miracidia, from BH and SJ strains respectively. Control snails of both species were maintained in dechlorinated tap water without saccharose and exposed to the same number of miracidia. There was no significant difference between the infection rates of snails treated or not with saccharose. However, the two groups of B. glabrata had significantly greater infection rates than the corresponding B. tenagophila groups. Molluscs treated with saccharose had a lower survival rate, with the greatest mortality occurring immediately before and at the beginning of cercariae release. Treatment with saccharose did not result in the release of more cercariae, but larvae from molluscs so treated showed a greater capacity to penetrate mouse skin, which was attributed to the greater energy supply during larval development in the mollusc.     

Schistosoma mansoni: histological localization of gelatinase in the preacetabular glands of cercariae

Proteolytic activity was demonstrated in secretions from the preacetabular glands of cercariae of Schistosoma mansoni. Thin gelatin film substrates were lysed by living cercariae stimulated to penetrate by application on the films of skin surface lipid. Lysis was directly related to number of cercariae, time, and temperature of incubation and pH of the medium. Gelatinase activity in unfixed frozen sections of cercariae incubated on the gelatin films was in the preacetabular glands which are the source of the secretion emptied into skin during penetration. Protease activity, therefore, appears to be related to penetration. The schistosome larvae which made the penetration attempt satisfied the accepted criteria for schistosomules, and therefore appeared to have transformed into schistosomules even though they did not successfully penetrate anything.     

Plagiorchis noblei (Plagiorchiidae) in Aedes aegypti: parasite acquisition and host mortality in trickle infections

The prevalence and intensity of experimental infections of Aedes aegypti with the digenean Plagiorchis noblei increased significantly with the level of trickle exposure to cercariae. Daily exposure to doses of 16 cercariae/day yielded a mean infection intensity of 13.0 metacercariae; doses of 1 cercaria/day resulted in only 2.4 metacercariae per infected mosquito larva. The prevalence of infection rose from 46% at an exposure of 1 cercaria/day to 99% at 16 cercariae/day. Host mortality rose concomitantly from 25% to 88%. Host mortality and parasite acquisition were independent of environmental temperatures (21-29 C), despite the fact that developmental times, and consequently the number of daily exposures, were more than 50% greater at the low end of the temperature scale. This may be attributable to low activity of mosquito larvae and the resulting decrease in the number of encounters with cercariae.     

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog <Emphasis Type="Italic">Polypedates cruciger</Emphasis>

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern.     

The Anthelmintic Drug Niclosamide Induces Apoptosis,Impairs Metastasis and Reduces Immunosuppressive Cells in Breast Cancer Model

Breast carcinoma is the most common female cancer with considerable metastatic potential. Discovery of new therapeutic approaches for treatment of metastatic breast cancer is still needed. Here, we reported our finding with niclosamide, an FDA approved anthelmintic drug. The potency of niclosamide on breast cancer was assessed in vitro and in vivo. In this investigation, we found that niclosamide showed a dramatic growth inhibition against breast cancer cell lines and induced apoptosis of 4T1 cells in a dose-dependent manner. Further, Western blot analysis demonstrated the occurrence of its apoptosis was associated with activation of Cleaved caspases-3, down-regulation of Bcl-2, Mcl-1 and Survivin. Moreover, niclosamide blocked breast cancer cells migration and invasion, and the reduction of phosphorylated STAT3^Tyr705, phosphorylated FAK^Tyr925 and phosphorylated Src^Tyr416 were also observed. Furthermore, in our animal experiments, intraperitoneal administration of 20 mg/kg/d niclosamide suppressed 4T1 tumor growth without detectable toxicity. Histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, VEGF-positive cells and microvessel density (MVD) and an increase in Cleaved caspase-3-positive cells upon niclosamide. Notably, niclosamide reduced the number of myeloid-derived suppressor cells (MDSCs) in tumor tissues and blocked formation of pulmonary metastases. Taken together, these results demonstrated that niclosamide may be a promising candidate for breast cancer.     

In vitro efficacy of new antiprotozoals against Philasterides dicentrarchi (Ciliophora, Scuticociliatida)

Philasterides dicentrarchi is a histiophagous ciliate that causes severe losses in turbot and sea bass farming. This study investigated the in vitro efficacy against P. dicentrarchi of 85 newly synthesized compounds and 12 commercial compounds, of which 2 are fluoroquinolones (norfloxacine and lomefloxacine) with known antibacterial activity. Seventeen of the newly synthesized compounds (2 naphthyridines, 2 pyridothienodiazines and 13 pyridothienotriazines) and the fluoroquinolone norfloxacin showed good activity. The most promising compound was the pyridothienotriazine 12k, with activity similar to that of the salicylanilides niclosamide and oxiclozanide (MLC 0.8 mg l(-1) in PBS, 1.5 mg l(-1) in seawater; MLC = minimum 24 h lethal concentration).