The demonstration of pre-hepatic immune response to Fasciola hepatica in the mouse.

Harness E., Hughes D. L. and Doy T. G. 1976. The demonstration of a pre-hepatic immune response to Fasciola hepatica in the mouse. International Journal for Parasitology6: 15–17. Two groups of mice were immunized with either one oral dose of 20 irradiated metacercariae per mouse or 2 such doses given 7 days apart. Twenty-one days after immunization these mice together with non immunized controls were orally dosed with 100 normal metacercariae. Two days later immature flukes were recovered from the peritoneal cavity and counted. The numbers of flukes recovered from both immunized groups of mice were significantly lower than those obtained from the controls; this is taken to indicate that a protective mechanism operates at the intestinal wall.     

On the viability of Fasciola gigantica metacercariae ingested by Lymnaea auricularia

The effect of ingestion by Lymnaea auricularia on the viability and infectivity of Fasciola gigantica metacercariae was studied. The cyst wall was unaffected by the snail's digestive processes. Two rabbits, each infected with 50 ingested metacercariae, died at 83 and 87 days post-infection. Eight and 10 immature flukes were recovered from the livers, indicating that the metacercariae had remained infective after passing through the intestine of the snail.     

Development of the large American liver fluke, Fascioloides magna, in white-tailed deer, cattle, and sheep.

The comparative development of Fascioloides magna in white-tailed deer, cattle and sheep has been studied. Flukes were recovered from 72% of 32 deer administered 40 to 500 metacercariae, from 82% of 11 cattle administered 10 to 500 metacercariae, and from 53% of 15 sheep administered 8 to 200 metacercariae. The percentage recovery of the flukes administered as metacercariae was 4.1% of 6,130 in deer, 5.7% of 2,510 in cattle, and 4.7% of 1,213 in sheep. Flukes were recovered only from livers of infected deer, while in cattle, 1 fluke was also found in the lungs of each of 2 animals. In sheep, all but 10 flukes were recovered from the livers; 6 were found in the lungs and 4 in the abdominal cavities. The black iron porphyrin pigment associated with F. magna infection was found to be most widespread in cattle and sheep, but was also a pathognomonic feature in deer. Growth of the fluke was similar in all 3 host species tested, but eggs were passed only from deer, the normal definitive host. In cattle, the eggs were retained in the liver, and F. magna was lethal to sheep before its own maturity was attained. In cattle and deer, flukes matured approximately 7 months after exposure, but immature migrating flukes were found 12 months after infection and apparently can remain in this retarded state for an undetermined period of time.     

Experimental Fascioloides magna infections of mule deer (Odocoileus hemionus hemionus).

Six mule deer (Odocoileus hemionus hemionus) and one white-tailed deer (Odocoileus virginianus), approximately 5-mo-old, each were inoculated orally with 500 metacercariae of Fascioloides magna. All mule deer died from liver fluke infection between 69 and 134 days (mean = 114, SE = 9.9) after inoculation. Between 38 and 326 immature F. magna (mean = 102, SE = 45.5) were recovered from each deer at necropsy. Flukes were present in livers, lungs, and free in pleural and peritoneal spaces. Infection was characterized by necrotizing hepatitis, fibrosing peritonitis and pleuritis, and hematin pigment accumulation in liver, lung, and many other internal organs. Eggs of F. magna first were detected in feces of the white-tailed deer 28 wk after inoculation, and weekly thereafter until the healthy deer was euthanized at 31 wk. At necropsy, 205 F. magna, including 12 encapsulated mature and 193 nonencapsulated immature flukes were recovered from liver, lungs, and free in abdominal and thoracic spaces of the white-tailed deer. Based on these results, F. magna may be fatal to mule deer within 5 mo of infection. Like domestic sheep and goats, mule deer may be highly susceptible to infection, and it is unlikely mule deer can survive infection with large numbers of F. magna.     

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.     

Fasciola hepatica: The effect of challenge by the subcutaneous or intraperitoneal route with living adult flukes in sensitized rats

When rats, sensitized either by subcutaneous implantation of adult F. hepatica or by a normal oral infection with F. hepatica metacercariae, were challenged by implanting adult flukes in the peritoneal cavity, 23% of these flukes were killed in rats sensitized by subcutaneous implantation and 71% in the rats sensitized by the oral route. In contrast neither of these sensitization routes were effective against subcutaneous challenge with adult fluke. Histological evidence suggested that about half of the dead flukes found were killed shortly after transfer and these flukes were surrounded with mononuclear cells. The remaining dead flukes appear to have died after becoming surrounded with a cyst. These latter flukes were surrounded by neutrophils and this cell type was very prominent in the cysts of sensitized rats.     

Fasciola hepatica: site of resistance to reinfection in cattle

The effective site of resistance to reinfection of cattle with Fasciola hepatica was examined by recovery of challenge flukes from either the liver or body cavity. Calves infected 18 or 26 weeks previously with F. hepatica showed levels of resistance to reinfection of 56 and 94%, respectively, as assessed by recovery of flukes from the liver 15-16 weeks after challenge. Plasma glutamate dehydrogenase (EC 1.4.1.3; GLDH) enzyme activity estimations revealed only a marginal increase in these latter resistant calves compared with previously naive controls, indicating minimal liver damage as a result of migrating flukes. By comparison, when immature challenge flukes were recovered from the body cavity 4 or 14 days after infection of corresponding previously infected or naive calves, there was no significant difference in numbers. It appears, therefore, that, in cattle, resistance mechanisms are effective against challenge flukes at or soon after penetration of the liver.     

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)     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

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.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Acanthoparyphium tyosenense: the discovery of human infection and identification of its source

Acanthoparyphium tyosenense Yamaguti, 1939 (Digenea: Echinostomatidae), was originally reported as an avian intestinal parasite; here, its presence is reported in 10 humans in the Republic of Korea. The patients were 9 adults aged 35-66 yr (males and females) and a young girl aged 7 yr residing in 2 coastal villages in Puan-gun, Chollabuk-do. The worms were recovered after treatment with praziquantel and purgation with magnesium salts. A total of 158 specimens (1-107 specimens/individual) was collected, together with varying numbers of other intestinal flukes. The patients had eaten various kinds of brackish water mollusks caught in an estuary near their villages. Five bivalves and a gastropod species suspected as sources of human infection were collected and examined. Two bivalves (Mactra veneriformis and Solen grandis) and the gastropod (Neverita bicolor) were found to be infected with the metacercariae of A. tyosenense; adult flukes were confirmed after the experimental infection of chicks. The results show that A. tyosenense infects humans and that brackish water mollusks are the source of human infection.     

Centrocestus formosanus (Digenea: Heterophyidae) encysted in the freshwater fish, Puntius brevis, from Lao PDR

The metacercariae of Centrocestus formosanus, a minute intestinal trematode of mammals and birds, were detected in the freshwater fish, Puntius brevis, from Vientiane Municipality, Lao PDR. The metacercariae were experimentally fed to mice, and adult flukes were recovered in their small intestines 7 days later. The adult flukes were morphologically characterized by having 32 (rarely 34) circumoral spines arranged in 2 alternative rows, a large bipartite seminal vesicle, an oval-shaped ovary, and an X-shaped excretory bladder. Based on these characters, the adults were identified as Centrocestus formosanus (Nishigori, 1924). The taxonomic significance of C. formosanus, in relation to a closely related species, C. caninus (Leiper, 1913), is briefly discussed. It has been first verified by adult worm recovery that C. formosanus is prevalent in Vientiane areas of Lao PDR, taking the freshwater fish, P. brevis, as a second intermediate host.     

The fate of Fasciola hepatica metacercariae following challenge infection of immune rats

Groups of rats, infected 7 weeks previously with Fasciola hepatica, together with appropriate control groups, were challenged either orally or intraperitoneally with 30 metacercariae. The mean worm recovery from the previously infected, orally challenged rats was significantly lower than from their respective controls (2.2 +/- 1.1 worms as opposed to 9.0 +/- 2.6). There was no significant difference in mean worm recovery from the previously infected, intraperitoneally challenged rats and their respective controls (5.3 +/- 3.2 worms as opposed to 6.2 +/- 1.9). Livers of the orally challenged group appeared to be largely free from secondary damage but considerable damage was evident in rats which received an intraperitioneal challenge. This evidence supports the view that the gut acts as an important barrier to metacercariae of a challenge infection. In a further experiment, young flukes were recovered from the gut, abdominal cavity and liver of immune and control rats 9, 18, 27, 36 and 45 h after oral challenge. It was found that fewer flukes successfully penetrated the guts of immune rats (3%) than those of uninfected controls (13%), again pointing to the gut as a barrier to metacercariae of a challenge infection. Protective mechanisms that may operate at the level of the gut are discussed.     

Susceptibility of rats to infection with Toxocara pteropodis

Infective eggs of Toxocara pteropodis were administered to Wistar rats via oral and parenteral routes. Third-stage larvae were recovered from the livers of suckling young 8 days after oral infection, and from livers and lungs after intraperitoneal or subcutaneous inoculation of eggs. These larvae were short-lived as none were found in suckling mice killed 2 weeks post-infection. Larvae were not recovered from tissues of rats aged 22 days or more when inoculated orally, indicating that refractoriness to infection develops rapidly with growth. Small numbers of larvae were recovered from the lungs of older rats 4 days after subcutaneous but not after oral inoculation. Adult male Buffalo and Fisher rats were also totally resistant to oral infection. Hence, rats differ from mice in their susceptibility to T. pteropodis.     

In vivo studies of the early, peritoneal, cellular and free radical response in rats infected with Fasciola hepatica by flow cytometric analysis

Early recruitment of the peritoneal cell population was observed during migration of newly excysted juvenile flukes. The peritoneal lavages were examined for T cells, cytotoxic NK cells (CNK) and free radicals production of rats at an early stage of infection by Fasciola hepatica. Male Sprague–Dawley rats were infected with 50 metacercariae of F. hepatica and non-infected controls were euthanized 2, 4 and 7 days post infection (d.p.i.), respectively. The peritoneal fluid of experimental animals was analyzed by flow cytometry to estimate cell phenotypes. The peritoneal areas were infiltrated by inflammatory cells, particularly from numerous neutrophils, eosinophils and CD4+ lymphocytes, which were significantly higher for infected rats than non-infected. CNK cells dominated in the peritoneal fluid of infected rats as early as 2 d.p.i. However, after 4 d.p.i. there was a decreased level of CNK cells which may indicate a change from a cytotoxic natural killer (NK) to a regulatory NK response. The challenged group generated very high in vivo levels of inducible nitric oxide (NO) from eosinophils. Superoxide expression was very high in macrophages and neutrophils compared to the uninfected control. In conclusion, our studies suggest that early F. hepatica infection could directly affect lymphoid cells and generate a high in vivo NO production by eosinophils in the peritoneal cavity. Moreover juvenile flukes could stimulate the macrophages and neutrophils to generate H₂O₂ radicals. The host parasite interactions resulting from immune response regulation by effector cells and immune evasion are discussed.     

Chronological observation of intestinal lesions of rats experimentally infected with Echinostoma hortense

Intestinal histopathological changes due to infection with Echinostoma hortense (Trematoda) were studied in rats after experimental infection with the metacercariae. The metacercariae were obtained from the tadpoles of Rana nigromaculata, a second intermediate host infected in the laboratory. Total 18 albino rats (Sprague-Dawley) were given 200 matacercariae each and sacrificed on the day 1, 3, 7, 11, 22 or 44 post-infection (PI). Segments of the small intestine at 1, 3, 5, 8 and 30 cm posterior to the pylorus (PTP) were resected and studied histopathologically. 1. The flukes were seen to have intruded into the intervillous space in the upper small intestine at early stages (1-3 days PI), however, they were located mainly in the intestinal lumen at later stages (7-44 days PI). The flukes were sucking and destroying the epithelial layers of villi with their oral and ventral suckers. 2. Histopathological changes of the intestine were recognizable in as early as 1-3 days after infection, and the changes became severer as the infection progressed. 3. The intestinal mucosa was histopathologically characterized by villous atrophy and crypt hyperplasia throughout the infection period. Major villous changes were blunting, fusion, severe destruction and loss of epithelial layers of villi. Villous/crypt (V/C) height ratio was remarkably reduced from 3:1 in controls to 1:1 in severely infected animals. In the stroma of villi, inflammatory cell infiltrations, vascular congestion, edema, and/or fibrosis were recognized. The goblet cells were increased in number after 11 days PI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal histopathology and in situ postures of Gymnophalloides seoi in experimentally infected mice

The intestinal histopathology and in situ postures of Gymnophalloides seoi (Digenea: Gymnophallidae) were studied using C3H/HeN and C57BL/6 mice as experimental hosts; the effects of immunosuppression were also observed. The metacercariae isolated from naturally infected oysters, 300 or 1,000 in number, were infected orally to each mouse, and the mice were killed at days 3-21 post-infection (PI). In immunocompetent (IC) mice, only a small number of flukes were found in the mucosa of the duodenum and jejunum during days 3-7 PI, with their large oral suckers pinching and sucking the root of villi. The intestinal mucosa showed mild villous atrophy, crypt hyperplasia, and inflammations in the villous stroma and crypt, with remarkable goblet cell hyperplasia. These mucosal changes were almost restored after days 14-21 PI. In immunosuppressed (IS) mice, displacement as well as complete loss of villi adjacent to the flukes was frequently encountered, otherwise the histopathology was generally mild, with minimal goblet cell hyperplasia. In these mice, numerous flukes were found, and it seemed that they were actively moving and rotating in situ. Several flukes were found to have invaded into the submucosa, almost facing the serosa. These results indicate that in IC mice the intestinal histopathology caused by G. seoi is generally mild, and the flukes do not penetrate beyond the mucosa, however, in IS mice, the flukes can cause severe destruction of neighboring villi, and some of them invade into the submucosa.     

Fasciola hepatica: Migration of newly excysted juveniles in resistant rats

Using transmission and scanning electron microscopy, the early migration of juvenile Fasciola hepatica was examined in naive and resistant rats. In naive rats, the migration of flukes to the peritoneal cavity was uneventful. In resistant rats, flukes were rapidly coated with antibody whilst still in the gut lumen and a proportion of the flukes were unable to penetrate the intestinal wall. Those that did penetrate were unharmed as they crossed the gut wall, but on entering the peritoneal cavity they were coated with antibody and host cells including eosinophils, neutrophils, macrophages, and mast cells. Eosinophils were seen degranulating onto the fluke surface, and this appeared to result in the erosion of the tegumental syncytium.     

The culture of Diplostomum spathaceum metacercariae on the chick chorioallantois

Metacercariae (2,828) obtained from the lens of naturally infected Aplodinotus grunniens were transplanted onto chorioallantoic membranes (CAM's) of 94 eggs (means 30/egg) 5 to 12 days old. Membranes were examined 2 to 8 days later and 381 flukes were recovered. Two hundred fifty-nine chorioallantoic-grown worms were transferred to 28 additional embryos 3 to 8 days after the initial inoculation. These eggs were examined 3 to 11 days later. Fifty-three serially transferred worms were recovered after 7 to 14 days on CAM's. Six stages of development were recognized: stage 1, immature; stage 2, genital rudiment; stage 3, testes; stage 4, follicular ovary; stage 5, vitellaria; stage 6, ovigerous. The intestinal ceca of many worms (except stage 1) contained ingested blood. Trematode eggs oviposited on CAM's were embryonated in tap water, and viable miracidia were observed in 15. No previous study has achieved gonadal development in Diplostomum spathaceum on the CAM. Furthermore, inasmuch as metacercaria matured and produced fertile eggs, this form is shown to be a useful model for the study of trematode differentiation. The development of metacercariae to ovigerous adults capable of producing viable miracidia suggests that the CAM shares characteristics with the intestine of the definitive host, a piscivorous bird.