Fasciola hepatica: role of developmental stages in the rat's resistance to challenge

Rats were sensitized by subcutaneous implantation of either metacercariae, 4 week-old juveniles, adult worms, or eggs of Fasciola hepatica and then challenged with 30 metacercariae 2 weeks later. Worm burdens were determined 8 weeks after challenge. Apart from adult worms, all implanted stages conferred a significant degree of protection on the recipients. The effectiveness of adult worm implants was not improved by using worms from different sources (sheep and cattle rather than rats) nor by extending the period of sensitization prior to challenge.     

Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.     

The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.     

Extraintestinal migration of Pharyngostomum cordatum metacercariae in experimental rodents

Extraintestinal migration patterns of Pharyngostomum cordatum (Digenea: Neodiplostomidae) were studied in experimental rodents such as mice, rats, and hamsters. When metacercariae isolated from grass snakes were infected orally to rodents, they penetrated the intestinal wall at days 2-3 post-infection (p.i.) and were discovered mainly in the diaphragm, intercostal muscles, and vital organ such as the lungs at days 7-28 p.i., without morphological changes. Interestingly, from several rodents which died suddenly at days 2-9 p.i., small to considerable numbers of metacercariae were found, not only in the lungs, but also in the heart and brain. Within the tissues, worms were freely motile until day 7 p.i., but later they were surrounded by host cells, and finally tissue cysts were formed. When metacercariae harvested from the snakes and intercostal muscles of rodents were infected orally to cats, they developed into adult flukes in the small intestine. The results show that P. cordatum undergoes considerable extraintestinal migration including the vital organs of its rodent hosts.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

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.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

Fasciola hepatica in rats: transfer of immunity by serum and cells from infected to F. hepatica naive animals

Immune, hyperimmune, and nonimmune serum samples were collected from inbred rats following 10 to 15 weeks of one [5 metacercariae (mc)/rat], two (5 mc followed by 30 mc/rat) or no (uninfected) exposure to Fasciola hepatica. Lymphoid cells also were collected from these donors. Inbred, naive rats in groups receiving immune serum, hyperimmune serum, nonimmune serum (serum control), immune cells, hyperimmune cells, and nonimmune cells (cell control) received intraperitoneally either a total of 20 ml of serum or a total of 3 x 10(8) viable lymphoid cells. A challenge infection of 30 mc/rat was administered orally at about the time of serum or cell transfer. The transfer of immunity was evaluated by examining recipient rats for parasites 4 and 8 weeks after challenge. Some hematological parameters and the precipitating antibody response of the recipients were monitored also. Hyperimmune serum, unlike immune serum, consistently provided a significant degree of protection in recipient rats. The precipitating antibody titre of this serum was higher than that obtained from the immune donor group. The importance of a second sensitization to obtain sufficiently potent serum was demonstrated. Lymphoid cells from infected donors did not consistently confer protection on recipients. Thus, the expression of protective immunity against F. hepatica seemed to be more dependent on the presence of antibodies than on cells. The hematological parameters of the recipients, in general, supported this observation. The precipitating-antibody response of protected rats was lower than that of unprotected animals following challenge, presumably because the development of fewer worms in the former provided less antigenic stimulation.     

Action of diethylcarbamazine citrate on protective immunity in rats infected with Nippostrongylus brasiliensis

Rats made immune to Nippostrongylus brasiliensis and treated with diethylcarbamazine citrate (DEC) orally (250 mg/kg X 6) exhibited significant suppression of functional immunity. Similarly, administration of compound 48/80 (100 micrograms/rat i.p.) made the immune rats susceptible to challenge infection. Treatment of rats, with 22-day infection with compound 48/80, histamine (20 mg/rat, per os), or L-histidine (20 mg/rat, orally s.c.) did not accelerate worm expulsion. A massive complement-dependent adherence of peritoneal cells (1 X 10(8], isolated from immune DEC-treated and untreated rats, to infective larvae (L3) was observed. Likewise, heavy congregation of normal peritoneal cells to larvae was noticed when the cells were incubated with sera obtained from immune, DEC-treated or untreated rats. The rats receiving mesenteric lymph node cells (125 X 10(6) i.v.) or sera (0.5 ml or 1 ml X 3 i.p.), obtained from immune DEC-treated rats and challenged with infective larvae developed 50% more worms than those which received cells or serum from untreated immune donors. DEC appears to cause suppression of functional immunity and worm expulsion is not histamine mediated.     

Trichinella spiralis: selective intestinal immune deviation in the rat

In rats, infections with 100-2000 Trichinella spiralis muscle larvae lead to a prompt immunity that is expressed in parasite expulsion within 14 days. Rats infected with more than 2000 larvae display impaired immunity with rejection delayed by 50% (7 days) or more. Suppression is selective for expulsive immunity as the antifecundity response of rats is directly proportional to dose and is expressed sooner in heavily infected subjects. Suppression of intestinal expulsive immunity was suggested by the fact that, with low doses (2000 larvae or less), worm rejection was inhibited by cortisone, whereas cortisone inhibited antifecundity but had no discernable effect on worm rejection in high-dose infections. Evidence for local immune deviation as opposed to systemic immunosuppression was obtained in experiments using parabiotic rats. When one partner was infected with 6000 worms and the other with 200, the rat infected with 200 parasites showed earlier rejection than was seen in single controls infected with 200 worms. The prolonged survival of high-dose adults was not accompanied by a change in the site of worm residence in the gut. Immunological parameters such as serum antibody levels, the number of activated cells or specific anti-T. spiralis lymphocytes in thoracic duct lymph were all increased in a dose-dependent manner. These experiments therefore demonstrate a novel autoprotective mechanism by which adult T. spiralis selectively reduce the expression of expulsive immunity in the gut.     

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.     

Immunization with infective larvae of Strongyloides ratti (Nematoda) exposed to microwave radiation

Microwaves have not been tested previously for possible application in producing immunogenic preparations of parasites. This study examines the immunizing capacity of microwave-irradiated, infective larvae of Strongyloides ratti in rats. Rats were inoculated subcutaneously with untreated, microwaved, or microwaved and homogenized larvae, or distilled water, and challenged with untreated larvae. Data were collected on egg production and worm number/rat during primary infections and on egg production, worm number/rat, worm size, and eggs in utero/worm following challenge. Our results demonstrated that microwaved, infective larvae (intact or homogenized) of S. ratti were immunogenic for rats, even though they were incapable of reaching the intestine and maturing to adult worms. The immunity elicited by exposure to microwaved larvae was characterized on challenge by a significant reduction in the number of eggs produced/worm, by the formation of perioral plugs, and by reductions in worm numbers and size. These results suggest that microwave radiation may provide a valuable new tool for parasitic vaccine production. In addition, we have demonstrated the occurrence of a feature of the immune response of rats to S. ratti that may have been overlooked previously; i.e., a gut-level response that was elicited by larvae, but manifested against adult worms in the intestine.     

Development of resistance to reinfection by Clonorchis sinensis in rats

We investigated the induction of resistance to Clonorchis sinensis infection by prior infection in rat and hamster models. Animals were challenged with C. sinensis metacercariae, then treated with praziquantel and reinfected. Worm recovery rate in reinfected animals was used to estimate resistance to reinfection. The determined resistance rates to reinfection in rats and hamsters were 97.7% and 10.3%, respectively. In rats, cure from the primary infection of C. sinensis increased resistant to reinfection, and the greater the worm burden and the longer the duration of primary infection, the higher was the resistance rate. For primary infection doses of 10, 40 and 100 metacercariae per rat, the resistance rates were 87.4%, 93.8% and 98.4%, respectively. The resistance rates in rats after 2 or 8-week primary infection were 78.7% and 95.3%, respectively. All worms recovered from reinfected rats were immature. When cured rats were administered with methylprednisolone, resistance to reinfection became impaired. These findings indicate that rats develop a high degree of resistance to reinfection by C. sinensis after cure. The growths and maturations of reinfected worms were also impaired.     

The protective effect of irradiated metacercariae of Fasciola gigantica against homologous challenge in sheep

Five lambs were each sensitized with 400 metacercariae of Fasciola gigantica irradiated at 3 kr gamma rays. Eight weeks later, they were each challenged together with 5 controls with 500 non-irradiated metacercariae. Eight weeks later, all of the animals were necropsied and the worm burdens determined. The lambs which received the sensitizing infections had 80% less worms than the controls. The sensitized lambs also showed less hepatic damage compared with the controls as indicated by lower levels of the serum enzyme glutamate dehydrogenase. The blood indices showed insignificant reductions in the sensitized lambs whereas marked reductions were found in the erythrocytes, packed cell volume and haemoglobin values of the challenge controls. Bilirubinaemia was also evident in this latter group.     

The reversibility of intestinal immune expulsion effects on adult Nippostrongylus brasiliensis

An attempt has been made to study the extent and nature of the damage occurring in adult Nippostrongylus brasiliensis undergoing immune expulsion from the rat. It was found that worms are not killed nor irreparably damaged when being rejected. On transfer into naive second recipient rats the rate of re-establishment of worms previously incubated in immune rat recipients for 4-17 hr was high (68-69%) and comparable to that shown by worms from normal recipient rats (48-56%). Similarly, worms taken on days 10, 11, and 12 of a primary infection, already passed to the distal half of the small intestine due to immune expulsion effects, on transfer into naive recipient rats re-established themselves well (rates varying from 62 to 80%) compared to those harvested from their normal habitat in the proximal half of the small intestine (rates varying from 44 to 87%). Worm damage is associated with decreased motility and impaired locomotion capacity. The phenomenon of mucosal trapping occurs during expulsion, but merely to the extent of some 30% of the worm population. It is suggested that in principle, worms subjected to immune expulsion are in a state of acute, transient metabolic crisis. The present results support the enteroallergic indirect mechanism for worm rejection.     

Animal Models for Echinostoma malayanum Infection: Worm Recovery and Some Pathology

Echinostomes are intestinal trematodes that infect a wide range of vertebrate hosts, including humans, in their adult stage and also parasitize numerous invertebrate and cold-blooded vertebrate hosts in their larval stages. The purpose of this study was to compare Echinostoma malayanum parasite growth, including worm recovery, body size of adult worms, eggs per worm, eggs per gram of feces, and pathological changes in the small intestine of experimental animals. In this study, 6-8-week-old male hamsters, rats, mice, and gerbils were infected with echinostome metacercariae and then sacrificed at day 60 post-infection. The small intestine and feces of each infected animal were collected and then processed for analysis. The results showed that worm recovery, eggs per worm, and eggs per gram of feces from all infected hamsters were higher compared with infected rats and mice. However, in infected gerbils, no parasites were observed in the small intestine, and there were no parasite eggs in the feces. The volume of eggs per gram of feces and eggs per worm were related to parasite size. The results of histopathological changes in the small intestine of infected groups showed abnormal villi and goblet cells, as evidenced by short villi and an increase in the number and size of goblet cells compared with the normal control group.     

Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat

Applicability of the adult Paragonimus westermani antigen for detection of anti-immature P. westermani antibodies in experimentally infected rats, a paratenic host of this lung fluke, was examined. The serum antibodies of the cats and rats infected with P. westermani metacercariae were detected by enzyme-linked immunosorbent assay (ELISA) with the adult-fluke antigen. The ELISA titers of serum samples of the rats infected with only immature flukes were as high as those of the cats infected with adult flukes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and the immunoblotting technique showed that a major protein band of 27,000 daltons was recognized in the sera of the infected cats and rats. Immunoperoxidase staining applied on the sectioned flukes provided evidence showing that the antigenic substance was located on the surface of the gut epithelium and in the luminal contents in both adult and immature flukes. The adult-fluke antigen containing the 27,000-dalton substance is applicable as a standard antigen for diagnosis of paragonimiasis westermani in not only definitive hosts but also in paratenic hosts.     

Immunization of zebu calves against Fasciola gigantica, using irradiated metacercariae

The pathogenesis of unirradiated, 3 krad-irradiated and 20 krad-irradiated metacercarial infections was compared in zebu calves studied over a 10-week period. Calves exposed to 1000 unirradiated metacercariae (mc) became hypoalbuminaemic, and showed elevated serum concentrations of liver enzymes, whereas neither of the other groups was significantly affected. At slaughter, a mean of 332 flukes was recovered from the 0 krad group, while only 23% and 12% of this number were recovered from the 3 krad and and 20 krad groups, respectively. All the worms recovered from the 20 krad group were stunted, and found in biliary ductules, but a mean of 13% of the flukes recovered from the 3 krad group were large, and dwelling in main bile-ducts. Liver lesions typical of acute fascioliasis were present in the 0 krad group, but lesions in the other groups, and particularly the 20 krad group, were far less severe. Judged on clinico-pathological criteria, a single vaccination of calves with 1000 3 krad-irradiated mc induced partial resistance to a challenge with 1000 normal mc eight weeks later, but the reduction in worm recovery was not statistically significant. There was less evidence of protection when two vaccinating doses of 3 krad mc were given within four weeks, with challenge at week 8, and a single vaccination was ineffective against a challenge four weeks later. However, when the irradiation dose was increased to 20 krad, a hgh level of resistance (69% worm reduction) was induced by a single vaccination, given eight weeks before challenge, and liver pathology was strikingly reduced in the vaccinated animals.