Schistosoma mansoni: Infectivity and immunizing effects of in vitro derived schistosomula attenuated by X irradiation

Irradiated and nonirradiated in vitro derived schistosomula of Schistosoma mansoni were injected intraperitoneally into mice. Sixteen percent of nonirradiated schistosomula, 8% of those irradiated with 1000 R, and virtually none of those irradiated with 3000 R and above survived in mice for 5 weeks. However, those irradiated with 3000 R survived in small numbers for shorter periods of time. Schistosomula irradiated with 3000 or 6000 R were used to immunize mice against subsequent infection with cercariae. Prior ip injections of schistosomula irradiated with 3000 R resulted in reductions in worm burden after challenge from 5 to 91%; the observed protection was related to the number of inoculations. The subcutaneous route appeared to be less effective. Schistosomula irradiated with 6000 R produced less protection than those irradiated with 3000 R.     

In vitro culture of Trichobilharzia ocellata

Cercariae and schistosomula of Trichobilharzia ocellata were cultured to the organogeny stage in vitro in a medium based on Earle's saline (gassed with 5% CO₂ in air) and containing lactalbumen hydrolysate and duck or chicken serum together with homologous red cells. Similar results were obtained irrespective of whether cercariae, schistosomula recovered from the skin of ducklings or chickens, or schistosomula recovered after cercariae had penetrated gelatine membranes were used to initiate cultures. A lipid coat developed around the body of each worm during the first 2–3 days in vitro, but subsequently it became dislodged. The worms ingested red cells, and the caeca became dark with haematin after a few days but by day 10 they were generally translucent. Maximum development was achieved at day 12; by this time males had attained a length of 2·1 mm and females 1·4 mm. Worms cultured for 7 and 9 days were injected into the leg veins of ducks and patent infections were established.     

Host-parasite relationships of Fasciola hepatica in the white mouse. VII. effects of anti-worm incubate sera on transferred worms and successful vaccination with a crude incubate antigen.

Mouse antisera against the 16-day-old worm incubate and sera from 25-day infections in mice debilitated migrating flukes in recipient animals as measured by worm recovery and host mortality. Mouse antisomatic and 100-day infection sera produced no such effects. Host mortality was significantly lower after challenge in mice given one ip immunizing injection of the worm incubate; however, worm recovery was not significantly reduced. Injections at 2, 7, 12, and 24 hr with the worm incubate elaborated over a 24-hr period protected 75% of the mice from infection after challenge, and reduced the worm burden by 83.3%.     

Schistosoma mansoni: Vaccination of mice with highly X-irradiated cercariae

Vaccination against schistosomiasis with highly X-irradiated Schistosoma mansoni cercariae was studied in mice. The optimum dose of X radiation for the attenuation of cercariae was in the range of 24–48 krad. In selecting the optimum dose, lesions caused by migrating schistosomula in the lungs of the immunized host were considered. Cercariae exposed to 48 krad caused fewer lesions than those exposed to 24 krad but still effected a comparable worm reduction. The percentages of worm reduction in mice immunized with 48-krad X-irradiated cercariae increased with the number of immunizations up to the fifth immunization and then fluctuated in the sixth, seventh, and eighth days without increase. The optimum dose of immunizing cercariae was 500, and the optimum time interval for successive immunizations was 4 weeks. There was no significant difference in susceptibility to infection in the adult mice 161 to 694 days of age. The duration of acquired immunity in immunized mice is long, still evident 545 days from the last immunization. The present studies clearly showed that with the bioengineering method, the worm reduction in the immunized mice reached 91.1%, the effect of immunization was stronger in mice immunized with the highly X-irradiated cercariae than with the low X-irradiated cercariae, and X-irradiated cercariae were demonstrated to be a strong inducing agent for immunity in mice.     

Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Schistosoma mansoni: reduced worm burdens in mice immunized with isolated Fasciola hepatica antigens.

Mice immunized with Fasciola hepatica antigens are protected to a challenge exposure with Schistosoma mansoni cercariae. This protection is manifested in a 28–54% reduction in worm burdens of the immunized mice over controls. The protective antigens could be isolated by antibody affinity chromatography and react with an antiserum to S. mansoni. These antigens, when used to immunize mice, result in 50–60% reduction in worm burdens over controls. One protective antigen has been isolated which when used alone or in combination with a B-cell adjuvant such as polyadenylic-polyuridylic acid (poly (AU)) results in 56–81% reduction in worm burdens over controls. The complexity of the F. hepatica adult worm antigens was demonstrated by Laurell crossed immunoelectrophoresis. Crossreactivity with antisera to S. mansoni and S. japonicum and the presence of one common antigen between the two genera have been demonstrated.     

Reappraisal of experimental infections with cercariae and schistosomula of a Brazilian strain of Schistosoma mansoni in mice.

The present investigation involves a reevaluation of previous results obtained after experimental infection of Swiss Webster mice with cercariae and schistosomula of the Schistosoma mansoni LE strain maintained under laboratory conditions. Three experimental groups of mice were considered: the animals of the first group were percutaneously (ring method) infected with cercariae, those of the second were subcutaneously inoculated with cercariae and the mice of the third were inoculated by the same route with schistosomula transformed in vitro. The data obtained so far indicated that the most effective method of infection is the subcutaneous injection with schistosomula, with a mean adult worm burden recovery of 54.1% when compared to the abdominal percutaneous and subcutaneous routes of infection with cercariae, in which the values were 36.7% and 32.4%, respectively. This suggests that, in experimental infections of SW mice with a LE S. mansoni strain, the skin is to be considered an effective attrition site in the percutaneous route, whereas in the case of inoculation with cercariae, a small amount of larvae fails to be transformed into viable schistosomula, possibly due to skin phase avoidance. A brief discussion about attrition sites and elimination of larval S. mansoni worms in mice is presented.     

Trichinella spiralis: dose dependence and kinetics of the mucosal immune response in mice.

The role of the mucosal immune response in helminth infections is not clear. In this study, the dose dependence and kinetics of the mucosal immune response to Trichinella spiralis were determined in experimentally infected Swiss Webster and BALB/c mice. The primary mucosal isotype was sIgA, although IgG was also detected, and primary infections with 10 and 150 larvae produced an anamnestic response on challenge. The mucosal and systemic immunoglobulin responses were dose dependent in both primary and challenge infections. The fecundity and length of worms and the rate of expulsion from the gut were determined on Day 6 postchallenge in Swiss Webster mice. Adult worm recovery and fecundity were reduced by greater than 50% and worm length by 28% in mice infected and challenged with 10 larvae and by 90, 85, and 35%, respectively, in mice infected and challenged with 150 larvae. The rate of expulsion was correlated with the size of both primary and challenge doses and a reduction in fecundity was correlated with the size of the primary dose only. The reduction in worm length did not differ significantly between the infection doses, but the trend was similar to that for expulsion. In BALB/c mice the expulsion response was dissociated from a reduction in fecundity and worm length, the latter two being positively correlated with sIgA levels, supporting a role for sIgA and/or IgG in these effects. However, expulsion does not appear to be dependent on the mucosal immunoglobulin response.     

Effects of host endocrine gland removal on the permissive status of laboratory rodents to infection by Schistosoma mansoni

Knopf P. M. and Soliman M. 1980. Effects of host endocrine gland removal on the permissive status of laboratory rodents to infection by Schistosoma mansoni. International Journal for Parasitology, 10: 197–204. The capacity of Schistosoma mansoni to complete its life cycle was compared in CD-1 mice (permissive hosts) and Sprague-Dawley rats (nonpermissive hosts) from which the pituitary gland had been removed prior to infection with cercariae. Except for a modest decrease in egg burden, none of the parameters of worm life cycle assessed were affected in hypophysectomized mice. In contrast, all these parameters were affected in hypophysectomized rats, e.g. onset of adult worm elimination was delayed, worm development improved, oviposition increased and miracidia developed. Effects of removal from rats of the thyroid/parathyroid glands on the parasite life cycle were similar to hypophysectomy; adrenalectomy or gonadectomy were without affect. Differences between thyroidectomized and thymectomized rats are discussed. It is concluded that host hormones contribute to the nonpermissive status of rats to Schistosoma mansoni infections.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice

Cross-resistance in Schistosoma mansoni and Fasciola hepatica infections were studied in mice. A primary infection of S. mansoni, 7 to 28 days old, did not stimulate a significant level of resistance to heterologous challenge with F. hepatica. In contrast, in older S. mansoni infections (54–65 days old) there was a significant level of resistance to a challenge with F. hepatica. The F. hepatica worm burden was reduced by 34.0 to 72.5% in separate experiments. Challenge infection with F. hepatica did not influence the number of S. mansoni in primary infections. No heterologous resistance to S. mansoni was found in mice with 7- and 23-day-old F. hepatica infections. However, primary infections with F. hepatica, 28, 32, 42, and 50 days old, conferred significant resistance to a heterologous challenge with S. mansoni. The established schistosome worm burden was reduced by 41.5 to 50.4%. In no case was the primary F. hepatica burden reciprocally influenced by challenge infection with S. mansoni.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

The influence of salinity on the cercariae of three species of Schistosoma

Donnelly F. A., Appleton C. C. and Schutte C. H. J. 1984. The influence of salinity on the cercariae of three species of Schistosoma. International Journal for Parasitology14: 13–21. The effect of salinity on the longevity and infectivity of cercariae of Schistosoma mattheei, Schistosoma haematobium and Schistosoma mansoni was determined. No significant differences in cercarial longevity occurred (p > 0.05) in low salinities (0–5.25%), whereas further increases in salinity resulted in progressive decreases in survival. In salinities ? 17.5%, cercariae were incapable of surviving for longer than 11 min. A maximum life-span of up to 122 h was recorded for some S. mattheei cercariae. Cercarial infectivity, as indicated by worm returns, was reduced progressively with increasing salinity up to a lethal limit of 10.5%. Differences in the salinity tolerance of the cercariae of the three species were discussed.     

Praziquantel-encapsulated niosomes against Schistosoma mansoni with reduced sensitivity to praziquantel

Introduction: Praziquantel (PZQ) is the only commercially available drug for schistosomiasis. The current shortage of alternative effective drugs and the lack of successful preventive measures enhance its value. The increase in the prevalence of PZQ resistance under sustained drug pressure is, therefore, an upcoming issue.Objective: To overcome the tolerance to PZQ using nanotechnology after laboratory induction of a Schistosoma mansoni isolate with reduced sensitivity to the drug during the intramolluscan phase.Materials and methods: Shedding snails were treated with PZQ doses of 200 mg/kg twice/ week followed by an interval of one week and then repeated twice in the same manner. The success of inducing reduced sensitivity was confirmed in vitro via the reduction of cercarial response to PZQ regarding their swimming activity and death percentage at different examination times.Results: Oral treatment with a single PZQ dose of 500 mg/kg in mice infected with cercariae with reduced sensitivity to PZQ revealed a non-significant reduction (35.1%) of total worm burden compared to non-treated control mice. Orally inoculated PZQ- encapsulated niosomes against S. mansoni with reduced sensitivity to PZQ successfully regained the pathogen’s sensitivity to PZQ as evidenced by measuring different parameters in comparison to the non-treated infected animals with parasites with reduced sensitivity to PZQ. The mean total worm load was 1.33 ± 0.52 with a statistically significant reduction of 94.09% and complete eradication of male worms. We obtained a remarkable increase in the percentage reduction of tissue egg counts in the liver and intestine (97.68% and 98.56%, respectively) associated with a massive increase in dead eggs and the complete absence of immature stages.Conclusion: PZQ-encapsulated niosomes restored the drug sensitivity against laboratory- induced S. mansoni adult worms with reduced sensitivity to PZQ.     

IL-22 Mediates Goblet Cell Hyperplasia and Worm Expulsion in Intestinal Helminth Infection

Type 2 immune responses are essential in protection against intestinal helminth infections. In this study we show that IL-22, a cytokine important in defence against bacterial infections in the intestinal tract, is also a critical mediator of anti-helminth immunity. After infection with Nippostrongylus brasiliensis, a rodent hookworm, IL-22-deficient mice showed impaired worm expulsion despite normal levels of type 2 cytokine production. The impaired worm expulsion correlated with reduced goblet cell hyperplasia and reduced expression of goblet cell markers. We further confirmed our findings in a second nematode model, the murine whipworm Trichuris muris. T.muris infected IL-22-deficient mice had a similar phenotype to that seen in N.brasiliensis infection, with impaired worm expulsion and reduced goblet cell hyperplasia. Ex vivo and in vitro analysis demonstrated that IL-22 is able to directly induce the expression of several goblet cell markers, including mucins. Taken together, our findings reveal that IL-22 plays an important role in goblet cell activation, and thus, a key role in anti-helminth immunity.     

Schistosoma japonicum: The design and experimental evaluation of a multivalent DNA vaccine

The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO₂SjFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO₂, pVIVO₂Sj23, pVIVO₂SjFABP and pVIVO₂SjFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO₂SjFABP-23, pVIVO₂Sj23 or pVIVO₂SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO₂SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.     

Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).     

Schistosoma mansoni: reduction in clinical manifestations and in worm burdens conferred by serum and transfer factor from immune or normal rhesus monkeys.

Although delayed hypersensitivity to Schistosoma mansoni was conferred on rhesus monkeys (Macaca mulatta) by means of dialyzable transfer factor prepared from peripheral leukocytes or lymph node cells of infected immune donors, when such animals were challenged with 1000 cercariae of S. mansoni they developed worm burdens similar to those of nontreated controls. However, recipients of transfer factor that, in addition, received hyperimmune serum showed minimal clinical symptoms and significantly reduced worm burdens after subsequent infection with S. mansoni irrespective of whether the donors used for the transfer factor were immune or uninfected. A significant but lower degree of protection was conferred by combinations of either S. mansoni transfer factor or normal transfer factor and normal serum. Neither transfer factor nor hyperimmune serum alone conferred protection to recipients. Susceptibility to infection was assessed by observing the signs of the disease, determining the worm burdens by perfusion 10 weeks after exposure, and by observing the appearance of the intestine at autopsy. The animals which received transfer factor and immune serum were protected against clinical disease. Good correlation between worm burdens and severity of disease was observed.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.