The role and immunophenotypic characteristics of myofibroblasts in liver of sheep naturally infected with the lancet liver fluke (Dicrocoelium dendriticum).

The main objective of our research was to examine the role and immunophenotypic characteristics of myofibroblasts in sheep liver naturally infected by the lancet liver fluke (Dicrocoelium dendriticum). In the reported study we analyzed liver samples from 20 adult sheep, 14 infected animals and 6 controls. The liver samples were fixed in 10% buffered formalin, and routinely processed and stained using hematoxylin eosin, the periodic acid-Schiff and Masson-Goldner trichrome methods. The immunohistochemical examination was carried out by the streptavidin biotin (LSAB2) method, using antibodies for α-smooth muscle actin (α-SMA), desmin and vimentin. The histopathological examination revealed liver fibrosis in 6 out of 14 (42.9%) analyzed samples, while different forms of cholangitis were observed in the remaining 8 out of 14 (57.1%). The expression of α-SMA was proven in perisinusoidal hepatic stellate cells, portal/septal myofibroblasts, and interface myofibroblasts. The degree of α-SMA expression and the number of α-SMA immunopositive cells were the most intensive in the liver with fibrosis. Desmin expression in all liver samples of infected sheep was confirmed in hepatic stellate cells and smooth muscle cells. The hepatic stellate cells, portal/septal myofibroblasts, and interface myofibroblasts reacted as vimentin positive cells. In the liver without fibrotic changes hepatic stellate cells and smooth muscle cells were desmin positive. The obtained results suggest that all populations of myofibroblasts, especially hepatic stellate cells, play an important role in the increased extracellular matrix formation during parasitic liver fibrosis in sheep naturally infected with D. dendriticum.

Dicrocoelium dendriticum in cattle from Cypress Hills, Canada: humoral response and preliminary evaluation of an ELISA.

Apparent changes in the distribution of Dicrocoelium dendriticum in western Canada and the absence of other liver flukes in the area prompted an examination of the humoral responses in naturally infected cattle sharing pasture with heavily infected wild cervids. Western blots using sera from the infected cattle indicated that the cattle had antibodies to various fluke antigens, however the pattern of response varied among isotypes. Isotype analysis indicated that Dicrocoelium-infected cattle produced IgG1, and IgM antibodies, but not IgG2 antibodies. Subsequent comparison of the efficiency of coprological and serological approaches for diagnosis showed that while 43.5% of a separate group of 26 yearlings had eggs in their faeces, the immunoblots indicated that all had detectable antibodies. A group of cattle necropsied for confirmation of fluke status was used to evaluate humoral immune responses and to provide baseline information in the preliminary development of an indirect ELISA for detection of anti-fluke antibodies.

Immunohistochemical study of the local immune response in lambs experimentally infected with Dicrocoelium dendriticum (Digenea).

Phenotypic expression of inflammatory cells in liver and hepatic lymph nodes (HLN) has been examined in lambs experimentally infected with Dicrocoelium dendriticum using immunohistochemical techniques. Thirty-two lambs, 12 infected with 1,000 D. dendriticum metacercariae, 12 with 3,000, and 8 controls were used. Half the lambs in each group were slaughtered on days 60 and 180 post-infection (p.i.), respectively. Primary antibodies (Abs) against T cell epitopes (CD3+, CD4+, CD8+ and WC1+ gammadelta), B cell epitopes (CD79alphacy+, CD45R+), immunoglobulin (IgG)-bearing plasma cells, macrophages (CD14+, VPM32+) and major histocompatibility complex (MHC) class IIbeta antigen were used. T lymphocytes (CD3+, CD4+ and CD8+) and B lymphocytes (CD79alphacy+ and CD45R+) with diffuse pattern or forming lymphoid aggregates and follicles surrounded the septal bile ducts (SBD) and inter-lobular bile ducts, whereas the WC1 gammadelta T cells were scattered. Numerous IgG+ plasma cells were observed around SBD. CD14 and VPM32+ macrophages intermingled with lymphocytes were immunostained by the anti-MHC class IIbeta. This Ab also reacted with lymphoid cells. Likewise, increased positive immunostaining for all Abs used was observed in the HLN of infected lambs. There was no qualitative difference regarding the phenotype expression of inflammatory cells between the lambs infected with D. dendriticum. The humoral and cell-mediated local immune responses observed were similar in the two groups of lambs infected with different doses.

Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliasis in northern Spain.

The transmission, control and the relationship between Dicrocoelium dendriticum and its definitive (sheep and cattle) and intermediate (molluscs and ants) hosts under natural and experimental conditions are described. Eleven species of molluscs and four of ants were found infected with larval D. dendriticum in León province, north-west Spain. Infected ants were observed between April and November and in tetania at 7.5-26.9 degrees C. The highest shedding of eggs by sheep and cattle was detected in winter. Two treatments applied in November and January were the most effective. In experimentally infected molluscs, the parasite was not visible under the stereomicroscope, at least until 50 days post-infection (p.i.). The prepatent period in experimentally infected lambs was 49-79 days p.i. The number of eggs per gram increased with the days p.i. and the parasite burden. The aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, leukocyte and neutrophil values of infected lambs increased, but those of lymphocytes decreased. Using the enzyme-linked immunosorbert assay technique, the IgG antibody response to excretory-secretory and somatic antigens of D. dendriticum was positive from day 30 p.i., although the maximum antibody levels were observed on day 60 p.i. The number of worms per lamb ranged between 30 and 2063. Cholangitis and cholangiectasia of the septal bile and hepatic ducts were observed. The best enzymatic systems for adult and larval D. dendriticum characterization were lactate dehydrogenase, glucose phosphate isomerase and phosphoglucomutase. Genetic variability of adult D. dendriticum was high using the random amplified polymorphic DNA technique.

Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy).

An indirect enzyme-linked immunosorbent assay (ELISA) with Dicrocoelium dendriticum excretory/secretory antigens was used to evaluate the presence of serum antibodies against the trematode in 738 sheep randomly chosen in Sardinia (Italy). Coprological sedimentation was used to discover egg-output. Seropositivity was detected in 86.2% tested sheep, whereas faecal prevalence was 6.7%; all that were faecal-positive also were ELISA-positive.

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle.

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.

Experimental dicrocoeliasis: the humoral immune response of golden hamsters and rabbits to primary infection with Dicrocoelium dendriticum.

The results presented deal with the humoral immune response of golden hamsters to primary experimental infection with D. dendriticum. The development of serum antibodies has been comparatively investigated with three hamster groups (n = 43) harbouring different burdens of adult flukes. The mean numbers of parasites were 11, 30, or 130 per animal. Serum antibody response was studied during an observation period of at least 331 and up to 496 days postinfection. For antibody detection the sensitivities of precipitation test (PTs) (double diffusion test, immuno- and counterimmunoelectrophoresis), of the indirect haemagglutination test (IHAT), the complement fixation test (CFT), and the enzyme linked immuno sorbent assay (ELISA) were compared using aqueous crude fluke antigen and crude egg antigen. CFT and ELISA were most sensitive for the early detection of initial response. Thereafter all the tests employed revealed increasing antibody titres, which in general remained at constant levels and persisted until the end of the observation period with the exception of CF-antibodies. In general fluke antigen was found to be more sensitive than egg antigen. However, in CFT this antigen occasionally has been associated with unspecific inhibition of haemolysis. Comparison of the results shows that ELISA using crude fluke antigen gave the most realistic picture of the actual fluke burden. Also preliminary results on the precipitin response of rabbits (n = 3) after primary experimental exposure to different numbers of metacercariae (500, 1,000, and 3,000 per animal respectively) are reported. Employing the above mentioned PTs a persisting antibody response could be demonstrated only after exposure to at least 3,000 infective larvae. The initial response was found on day 63, the observation period was 550 days.