A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis

Davtes C. 1980. A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis. International Journal for Parasitology10: 217–266. The ultrastructure of in vitro cultured adults of Microphallus similis was examined by TEM and SEM and compared to that of metacercariae and in vivo grown adults. In cultured flukes the most conspicuous abnormalities were observed in secretory cells, especially those of the tegument, digestive caecum, forebody glands and vitellaria. In the tegumental cells and the forebody gland cells, the secretory granules appeared to lose some of their contents within the cell bodies suggesting that there may be some defect in the transport and/or packaging of secretory granules in vitro. In the vitellaria of cultured flukes some of the granules lost their characteristic appearance, becoming ragged in outline and very electrondense. The premature tanning of the vitelline secretions within the vitellaria is correlated with abnormal egg production in vitro. The caecal cells of cultured flukes differed from those of the metacercaria and the normal adult in several important respects which suggested that their function was probably impaired.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Evidence for two distinct mechanisms of resistance in the rat to reinfection with Fasciola hepatica

Doy T. G. and Hughes D. L. 1982. Evidence for two distinct mechanisms of resistance in the rat to reinfection with Fasciola hepatica. International Journal for Parasitology12: 357–361. Congenitally athymic nude (rnu/rnu) and heterozygous hairy (rnu/ + ) rats were found to be equally highly resistant to oral reinfection with Fasciola hepatica. Accompanying the development of this resistance was a marked increase in intestinal eosinophil numbers. The sensitised rnu/ + rats showed a similar marked resistance to intraperitoneal challenge with newly excysted juvenile (NEJ) flukes. This was much less effective in the rnu/rnu rats, although there was some evidence of reduced numbers and stunting of parasites. Serum from infected rnu/rnu rats, unlike that from the infected rnu/+ rats, failed to induce the adherence of eosinophils to NEJ flukes in vitro. Flukes recovered from rnu/rnu rats were in general considerably larger than comparable flukes from their rnu/ + counterparts.There thus appears to be two distinct mechanisms of resistance to reinfection with F. hepatica operating in the rat. The first a T-independent system effective at the gut wall and the second, effective after penetration of the gut and requiring a T-dependent response for full expression. If eosinophils are involved in protection they can apparently function in the gut wall in the absence of an adherence promoting antibody in the serum.     

Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

In vitro cultivation of Fasciola hepatica metacercariae and of partially developed flukes recovered from mice

Attempts were made to culture the metacercariae of Fasciola hepatica under a wide variety of conditions. Of the media tested, the most successful was NCTC 135 plus 50% heat inactivated chick serum and sheep red blood cells at 37°–38°C. In this medium, somatic development of newly excysted juveniles was similar to that of flukes recovered from the liver of a mouse 11 days post-infection. There was, however, no corresponding development of the genital rudiment. Various supplements, such as liver extract, bile, yeast extract, embryo extract, egg products, monolayer cells and diphasic media were tested, but none enhanced development. The effects of various physical parameters on growth and development in vitro were examined. Cultured metacercariae appeared to be in a state of ‘suspended animation’; when injected intraperitoneally into mice they developed into egg-producing adults. Flukes recovered from the abdomen and liver of mice continued their somatic growth in vitro but their genitalia failed to develop further.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity

The response of newly excysted juvenile Fasciola hepatica to immune sheep serum under in vitro conditions was examined using indirect fluorescent antibody labeling and electron microscopy. Flukes acquired a continuous layer of host IgG over the surface during incubation in the presence of antiserum, but when transferred to a medium lacking antiserum they actively sloughed this layer and replaced the former glycocalyx, by a new antigenically similar surface coat. Electron microscope examination of juvenile flukes verified than an immune complex formed at and sloughed from the tegumental surface of those which were incubated in immune serum. T0 secretory bodies produced by the GER/Golgi system of the tegumental cells and stored in the metacercariae were discharged at the apical surface of the tegument, possibly in response to antibody binding. When cycloheximide was included with immune serum in the incubation medium the tegumental cells were unable to synthesize new T0 bodies to replace losses and the number of T0 bodies decreased so that the cytoplasm of the tegumental cells and surface syncytium became virtually devoid of T0 bodies within 48 hr.     

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.     

Further observations on the nature and characteristics of cross protection against Fasciola hepatica produced in sheep by infection with Cysticercus tenuicollis

Dineen J. K., Kelly J. D. and Campbell N. J. 1978. Further observations on the nature and characteristics of cross protection against Fasciola hepatica produced in sheep by infection with Cysticercus tenuicollis. International Journal for Parasitology8: 173–176. Previous studies showed that sheep infected with Cysticercus tenuicollis were protected against a subsequent infection with Fasciola hepatica given at 12 weeks (Campbell, Kelly. Townsend & Dineen, 1977). The present studies showed that these animals were again protected against re-challenge with F. hepatica at 9 months. Resistance was detected about 10 weeks after re-challenge with metacercariae.Sheep in which the initial C. tenuicollis infections were terminated by anthelmintic at 12 weeks, were resistant to the primary infection with F. hepatica but became fully susceptible to the re-challenge at 9 months.These results suggest that maintenance of resistance depends upon persistence of the C. tenuicollis infections. They also indicate that resistance is maintained by cysts in the peritoneum which is remote from the reactive site (liver).Infection with F. hepatica at 3 weeks after infection with C. tenuicollis prevented cross protection against both the primary infection with F. hepatica and re-challenge at 9 months.     

Age-associated responses in susceptible and resistant rats to infection with Fasciola hepatica

Rajasekariah G. R. and Howell M. J. 1981. Age-associated responses in susceptible and resistant rats to infection with Fasciola hepatica. International Journal for Parasitology11: 59–65. Groups of susceptible (5-week-old) and age resistant (25-week-old) outbred male Wistar rats were infected with metacercariae of Fasciola hepatica and the establishment of the parasite was assessed in terms of worm reocvery, and haematological, histopathological and immunological criteria, 2, 4, 6 and 8 weeks after infection. Apart from 2 weeks after infection, there was a significant difference between groups in the recovery of F. hepatica, with resistant rats infected with consistently fewer parasites than susceptible animals. The juvenile worms which invaded the livers of resistant rats elicited a number of host reactions, marked by an intensive cellular infiltration into migratory tracks of the parasite, heavy deposition of fibrous tissue in the liver parenchyma and a rapid antibody response. These responses were not as striking in susceptible animals even though more worms were present. The ability of resistant rats to mount an enhanced response seems related to the maturation of their haemopoietic system.     

The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Fasciola hepatica: Effects of diamfenetide free amine on in vitro physiology,biochemistry, and morphology

Short-term (1–3 hr) incubations in vitro of immature and adult Fasciola hepatica with 10^?4 to 10^?5M free amine of diamfenetide (DPT-FA) demonstrated a time/dose-dependent, irreversible paralysis that involved an increase in muscular tension and decrease in contraction amplitude. The following events occurred preceding or concomitant with the paralysis: influx of Na⁺, decrease in surface membrane potential, increase in wet weight, swellings on the ventral surface, and inhibition of 3-O-methyl glucose transport. These events were all consistent with a disturbance in surface membrane functions. The effects of DPT-FA were more severe in immature flukes (3–5 weeks postinfection) than adults which agrees with observed in vivo efficacy.     

Fasciola hepatica: Comparative studies on fascioliasis in rats and mice

Chapman C. B. and Mitchell G. F. 1982. Fasciola hepatica: comparative studies on fascioliasis in rats and mice. International Journal for Parasitology12: 81–91. Certain characteristics of infection differ between rats and mice exposed to metacercariae of the trematode parasite, Fasciola hepatica. Rats develop a degree of age-related resistance (and infected older females contain fewer parasites than older males), resistance to reinfection in infected rats is demonstrated readily though is partial, and a comparable degree of resistance can be obtained in recipients of infected rat serum provided the serum is given at about the time of challenge. None of these features of F. hepatica infection is seen in mice. Rats also differ from mice in that they can be vaccinated against infection (although again, resistance is incomplete) using larval antigen mixtures in adjuvants. Mice do respond to infection by production of antilarval antibodies and a slight IgG₁ hypergammaglobulinaemia and larvae will sensitize mice for delayed hypersensitivity. The results of this study indicate that sera from infected rats versus infected mice will be useful in pinpointing antigens of F. hepatica larvae which are involved in expression of partial host protection.     

In vitro development of the strobilar stage of Mesocestoides corti

Thompson R. C. A., Jue Sue L. P. and Buckley S. J. 1982. In vitro development of the strobilar stage of Mesocestoides corti. International Journal for Parasitology12: 303–314. Sexually mature strobilated adults of Mesocestoides corti were grown consistently from undifferentiated tetrathyridia in vitro using a conventional diphasic culture system. Development (growth, strobilisation and maturation) was compared in vitro and in vivo. Although growth and strobilisation were comparable in vitro and in vivo, during the first 18 days, total length and numbers of proglottids decreased in vivo but continued to increase in vitro after day 18. Both male and female reproductive systems appeared to develop normally in vitro and self copulation was frequently observed in cultured worms. However, fully developed oncospheres were not produced in vitro.     

Fasciola hepatica: Histochemical observations on juveniles and adults and the cytopathological changes induced in infected mouse liver

The histochemical characteristics of juvenile intrahepatic forms of Fasciola hepatica have been compared with those of the adults. The histochemical distributions of carboxylesterase (EC 3.1.1.1), acetylcholinesterase (EC 3.1.1.7) and alkaline phosphatase (EC 3.1.3.1) were similar in both juvenile and adult forms. Differences were apparent in occurrence and distribution of β-glucuronidase (EC 3.2.1.31) and N-acetyl-β-glucosaminidase (EC 3.2.1.29) activities in the tegument, parenchyma, and caecal cells. These may reflect the dietary mode and relationship to the host of the juvenile intrahepatic and adult bile-duct forms. Starvation of both juveniles and adults is accompanied by an increase in the granule-associated staining reactions for acid hydrolases in some of the parenchymal cells with a concomitant decrease in staining for glycogen in these cells. This response to starvation is reversible with refeeding, indicating that it is probably a genuine response to nutrient stress and not to degeneration induced by the in vitro conditions of the flukes. Carboxylesterase and acid phosphatase (EC 3.1.3.2) have been demonstrated to be polymorphic, using polyacrylamide disc electrophoresis. Changes were observed in the staining patterns with starvation. The juvenile flukes stimulated a considerable increase in the level of lysosomal hydrolases and alkaline phosphatase in mouse hepatic cells.     

Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae)

Mtitchell J. S., Halton D. W. and Smyth J. D. 1978. Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae). International Journal for Parasitology8: 389–397. Cotylurus erraticus metacercariae obtained from around the heart of rainbow trout were excysted and grown in vitro and in vivo to egg-producing adults. For in vitro development, tissue culture media M199 or NCTC 135 was used, together with varying amounts of chicken serum. Worms grown in media containing the highest concentration of serum (80% per volume) showed the fastest rate of development, measured by the time taken for the first eggs to appear in the uterus. The testes, ovaries and vitellaria of these worms were comparable in structure and histochemistry with those of worms reared in gulls. Eggs were produced by worms in all media containing chicken serum, but the eggs had abnormal shells and failed to embryonate.     

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: influence of extrahepatic adult flukes on infections and immunity in rats

Surgical transfer of adult Fasciola hepatica from sheep, goats, and cattle to subcutis of rats 4 wk before infection with metacercariae resulted in a 50% decrease in infection rate as compared to nonoperated controls.Infection was established in 25 out of 77 rats with F. hepatica implants, while 54 out of 79 were infected in the control group. The protective effect of the fluke implantation is discussed. It is suggested that production of protective antibodies is stimulated by the undamaged living flukes, although the antigen itself has not been demonstrated.     

Substantial genetic divergence between morphologically indistinguishable populations of Fasciola suggests the possibility of cryptic speciation

The liver flukes, Fasciola hepatica and Fasciola gigantica, are considered to be sister species and between them present a major threat worldwide to livestock production. In this study sequence data have been employed from informative regions of the nuclear and mitochondrial genomes of over 200 morphologically F. hepatica-like or F. gigantica-like flukes from Europe, sub-Saharan Africa and South Asia to assess genetic diversity. Evidence is presented for the existence of four well-separated clades: African gigantica-like flukes, Indian gigantica-like flukes, European hepatica-like flukes and African high-altitude hepatica-like flukes. Application of the Biological Species Concept to trematodes is problematic; however, the degree of separation between these groups was sufficient for them to be considered as distinct species using the four times rule for speciation.