An experimental study on triclabendazole resistance of Fasciola hepatica in sheep

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted.The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.     

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.     

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis.

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.     

Characterization of the humoral immune response in alpacas (Lama pacos) experimentally infected with Fasciola hepatica against cysteine proteinases Fas1 and Fas2 and histopathological findings

A characterization of the humoral immune response of alpacas to Fasciola hepatica Fas1 and Fas2 antigens, two abundant cysteine proteinases in the excretory/secretory (E/S) products, was performed over the course of 6 months of experimental infection. Six adult alpacas aged 1-2 years old received a single dose of 200 F. hepatica metacercariae; two non-infected alpacas were kept as control group. All infected animals shed eggs 8 weeks post-infection (PI) and the number of flukes recovered at necropsy averaged 41+/-4. The livers of infected animals showed regions with chronic inflammation, granuloma containing parasite eggs, necrosis and cirrhosis. Peripheral eosinophilia in infected animals was greatly enhanced 6 weeks post-infection and later. A single peak of serum glutamic piruvic transaminase (SGPT) was observed 4 weeks PI and serum glutamic oxalacetic transaminase (SGOT) elevated 3 weeks PI and later. Circulating IgG Abs against Fas1 and Fas2 were measured by enzyme-linked immunosorbent assay (ELISA). Fas2-ELISA detected the infection 10 days PI reaching to highest titer on 7-8 weeks PI and kept elevated, until the end of infection. Fas1-ELISA detected the infection 2 weeks PI and followed the same pattern as Fas2-ELISA. Anti Fas2 IgG Abs were in higher titers and showed stronger avidity than anti Fas1 IgG Abs. In addition, rabbit IgG antibodies raised against cysteine proteinase Fas2 showed infiltration of this parasite antigen associated to the degradation of bile ducts and liver parenchyma of infected alpacas. In the present study we have established a F. hepatica experimental infection of alpacas, Fas2 appears to have a role in the pathogenesis of the liver damage in alpacas caused by the liver fluke. Infected alpacas elicited a strong humoral immune response against fluke cysteine proteinases Fas1 and Fas2, which might be considered as candidates for immunodiagnosis and vaccine development against fasciolosis in alpacas.     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

Immune responses of cattle to experimental anti-Fasciola hepatica vaccines.

Fasciola hepatica infection of cattle and sheep is an important cause of clinical disease and production losses, and is controlled at present by a combination of chemotherapy and management measures. However, the prospects for the control of F. hepatica infection by vaccination are good, and we have previously shown substantial protection of cattle against experimental challenge infection following immunisation with a combination of the purified fluke-derived enzymes cathepsin L1 (CATL 1), cathepsin L2 (CATL 2) and fluke-derived Hb fraction (FHB). This and other recent studies have also demonstrated fundamental differences between protective and non-protective immune responses to liver fluke infection. In this present study we have further analysed the response of animals to liver fluke challenge following experimental vaccination. Calves were vaccinated with either CATL 2 plus FHB, or CATL 1 plus CATL 2. Partial protection against challenge infection was achieved in both vaccinated groups, with the greatest level of protection (55 per cent reduction in fluke burdens) recorded in the group vaccinated with CATL 1 plus CATL 2. This latter group also showed the greater level of lymphocyte proliferation and the greater production of gamma-INF in response to stimulation with fluke antigen in vitro following challenge. These results are significant in our attempts to characterise the elements within the immune response to vaccination which are protective.     

Cell-mediated immune response in calves to single-dose, trickle, and challenge infections with Fasciola hepatica

A peripheral blood mononuclear cell (PBMC) proliferation assay was used to study the cell-mediated immune response in eight calves experimentally infected with Fasciola hepatica. Hypersensitivity-related eosinophil and mast-cell responses were also assessed. The primary infection of 500 metacercariae was administered either as a single-dose or as a trickle infection over a 4-week period. Calves were challenge-infected 4 months later with 100 metacercariae and slaughtered 24 weeks postprimary infection. Skin eosinophil counts (SEC) were determined prior to infection on the basis of the intradermal reaction (IDR) to phytohaemagglutinin (PHA). These counts correlated negatively with the mean fluke length but not with the fluke burden found at necropsy. At the end of the experiment, non-specific (PHA) and specific (excretory-secretory parasite, products, FhESAg, and whole-worm extract, FhSomAg) immediate type hypersensitivity IDR were elicited in contrast to delayed type hypersensitivity (DTH) responses. The SEC correlated with blood eosinophilia but not with parasite parameters. These findings suggest that the eosinophil response does not correlate clearly with the development of resistance to F. hepatica infection in cattle. A specific mononuclear cell response to FhSomAg was detectable as early as 7 days after infection in both infected groups, being significantly higher during the very early migratory phase of the juveniles in the single-dose infected calves than in the trickle infected calves. This response remained significantly higher in infected groups than in the control group throughout the experiment. Challenge elicited a significant proliferative response, less pronounced than after primary infection. No production of gamma-interferon (INF-gamma) was recorded 3 weeks after challenge. At necropsy, the mean number of flukes recovered was similar in both infected groups, suggesting that the rate at which the infection is administrated has no effect on protective immunity. Hepatic lesions, similar in both infected groups, were characterised by marked eosinophil and mast-cell infiltration. Liver biopsies were performed and their diagnostic value is discussed. All results suggest that F. hepatica infection predominantly induces a Type-2 response in cattle, and that this response has little protective effect.     

Failure of a recombinant Schistosoma bovis-derived glutathione S-transferase to protect cattle against experimental Fasciola hepatica infection

The potential of a recombinant Schistosoma bovis 28-kDa glutathione S-transferase (rSb28GST) to protect cattle against Fasciola hepatica was tested in a vaccination trial. Thirty two calves were randomly divided into four groups of eight animals. Calves of the three vaccine groups received two intramuscular injections at 3 weeks interval, of 0.250mg rSb28GST in either aluminium hydroxide (Al(OH)(3)), Quil A, or PBS emulsified in an equal volume of Freund's complete adjuvant (FCA).Animals of the control group received injections of Al(OH)(3)/PBS only. All animals were challenged orally with a total of 360 metacercariae of F. hepatica, spread over 6 weeks.All groups of vaccinated animals produced measurable IgG antibody titers to rSb28GST after vaccination. Animals immunised with FCA adjuvanted vaccine had the highest and more durable antibody titers and only sera from this group recognised an approximately 24kDa protein band from F. hepatica, that is thought to be a F. hepatica GST. Despite a good antibody response differences in cumulative faecal egg output between the groups were not statistically significant. In addition, no significant difference was found between groups in terms of total worm numbers or percentage of immature flukes recovered at necropsy. In conclusion, the recombinant S. bovis 28kDa GST was not found to adequately protect cattle against experimental F. hepatica challenge, using either aluminium hydroxide, Quil A or FCA as adjuvant.     

The effect of pre-exposure to Fasciola hepatica or Schistosoma mansoni on challenge infection with Fasciola hepatica

Two groups of 12 and 6 rats were inoculated with Fasciola hepatica and Schistosoma mansoni, respectively. The Schistosoma-inoculated group, as well as 6 Fasciola-inoculated rats and 6 uninfected rats were challenged 8 weeks later with F. hepatica. A control group of 6 rats was left unexposed. Eight weeks after the challenge exposure all rats were necropsied and subjected to post-mortem examination. The number of Fasciola recovered after challenge was lower in both groups of rats primarily infected with F. hepatica or S. mansoni. F. hepatica-induced pathological changes were observed in all infected rats, but were pronounced in the group which was first exposed at the time of challenge of the primarily infected groups. No Schistosoma eggs or adults were detected in Schistosoma-inoculated rats. The results demonstrated that rats primarily infected with F. hepatica acquired resistance against a challenge exposure to the homologous parasite. Also S. mansoni, even without patency, can provide partial protection against F. hepatica infection.     

The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica

Fasciola hepatica causes significant morbidity and mortality in dairy cattle in the Andean region of Cajamarca, Peru, where prevalence of infection of up to 78% has been reported. ELISA and Western blot analyses were used to characterise antibody responses in dairy cattle to adult F. hepatica to excretory-secretory (E/S), somatic (SO) and surface (SU) antigens. Three groups of dairy cattle - calves, heifers and adult cows - naturally exposed to F. hepatica in this region, were monitored every 2 months over a 2-year period. Calves, heifers and adult cows all had antibodies which recognised a 28kDa protein in the SO preparation, whereas only adult cows had antibodies that recognised a 28kDa protein in E/S products. All three groups of cattle responded to a 60-66kDa group of proteins in E/S and SU preparations and a 17kDa antigen in SO products was recognised by antibodies from cows and heifers but not calves. The total antibody response to E/S antigens measured by ELISA, increased over time in calves and remained constantly high over the 2-year period in all three groups of cattle. Slight fluctuations in the antibody response occurred in the group of heifers and cows coinciding with seasonal changes in the level of challenge.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

Liver pathology and immune response in experimental Fasciola hepatica infections of goats

The relationship between the immune response and the pathogenesis of the disease was studied in different primary and secondary experimental Fasciola hepatica infections of goats. The establishment of the infection, measured as percentage of recovered flukes at the necropsy, was similar in primarily and secondarily infected animals (between 19.7% and 24.3%), but the hepatic damage was much more severe in secondarily infected goats, as revealed by the levels of serum hepatic enzymes GGT and LDH. Primary infection evolves to chronic fasciolosis that did not induce the development of resistance, since goats were highly susceptible to secondary infection, showing severe acute and chronic hepatic lesions that led to the death of some animals in each group. The immune response to the infection was proved by the production of specific IgG antibodies to ESP of F. hepatica and the involvement of CD3+ T lymphocytes and lambda IgG+ plasma cells in the hepatic infiltrate. Secondary infection did not induce any difference in either IgG response or in the cellular composition of the infiltrate of hepatic lesions, although this was much more extended. However, neither antibodies nor cell-mediated response were protective: there was no correlation between IgG levels and fluke burden and there was no evidence of cell-mediated killing of the parasite. This suggests the existence of some immune evasion mechanisms in goat infection with F. hepatica. The parasite may depress the local inflammatory and immune response, as suggested by the scarcity of CD3+ T cells in the infiltrate surrounding acute migratory tunnels. Moreover, in secondary infected goats can be suspected an immunological damage of the liver, since a very severe infiltrate of immune cells replaced wide areas of hepatic parenchyma and an immune-mediated damage of hepatocytes could occur.     

Vaccine potential of inclusion bodies containing cysteine proteinase of Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the fluke

Despite intensive research efforts, progress in the development of effective anti-Fasciola hepatica vaccine has not been satisfactory. However, it has been found that cysteine proteinases of F. hepatica are very important candidates for a vaccine antigen because of their role in fluke biology and in the host-parasite relationship. In our previous experiments we found that recombinant cysteine proteinase which we have cloned from adult F. hepatica (CPFhW) can protect rats against the liver fluke infection when administered intramuscularly or when given intranasally in the form of cDNA. In the present experiments we aimed to evaluate the protectivity of the mucosal vaccination in calves and lambs with inclusion bodies containing recombinant CPFhW using different vaccination doses and various sites of antigen delivery. Female calves vaccinated intranasally with two doses of 300 microg of the recombinant CPFhW showed 54.2% protection against the subsequent challenge of 400 metacercariae (mc). Flukes which developed in vaccinated calves showed a reduction of reproductive potential. Male Corriedale lambs vaccinated at the age of 4 months demanded three doses of the antigen to gain 56.5% of protection to a challenge with 250 mc of F. hepatica. Vaccinated animals showed significantly lower blood eosinophil counts. No correlation was found between serum and mucosal IgG or IgA reacting with F. hepatica ES antigens and the protection level.     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung

The potential synergistic effect of bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica in the production of pneumonia after aerosol/intranasal infection of conventionally reared lambs was evaluated. A mild clinical response was observed in lambs given virus and/or bacteria. Gross pulmonary lesions were seen in 3 of 6 lambs given RSV and then P haemolytica 3 or 6 days later, respectively (groups D and E), and in 1 lamb of 5 given virus and bacteria simultaneously (group G). Gross lesions were not seen in control sheep (group A), in lambs given virus or bacteria alone (groups B and C), or in lambs exposed to bacteria and then virus 3 days later (group F). Bovine RSV and P haemolytica were recovered from the lungs of 5 of 7 lambs with macroscopic lesions. Gross pulmonary lesions were cranioventral firm areas of red consolidation. Microscopically, the predominant lesion was a suppurative bronchopneumonia. Bovine RSV was recovered from the nasal cavity of 8 of 27 (30%) lambs given RSV during days 3 to 6 after viral inoculation, including 1 lamb in group B, 2 in groups D, E, and F, and 1 in group G. Pasteurella haemolytica was recovered from the nasal cavity of 9 of 28 (32%) inoculated lambs, including 2 lambs from groups C and E, 3 in group D, and 1 in groups F and G. Viral antigen, as determined by immunofluorescence, was concentrated mainly in individual cells in alveolar walls, some alveolar macrophages, and a few bronchiolar epithelial cells. In vitro alveolar macrophage assays indicated decreased numbers of Fc receptors on those macrophages collected from lambs given RSV 6 days before P haemolytica infection, as compared with that in the other groups. These cellular defects disappeared after 24 hours of culture. Seemingly, bovine RSV does facilitate P haemolytica pulmonary infection in conventional, immuno-competent lambs and provides evidence for decreased Fc receptors on alveolar macrophages.     

水牛感染肝片吸虫后肝脏组织的病理学变化

15 头水牛在人工感染条件下进行肝片吸虫感染, 慢性感染组水牛每天口服60 个囊蚴, 20 d 总共口服1 200 个囊蚴。急性感染组水牛1 次口服1 600 个囊蚴。慢性感染的水牛于感染后第26 周宰杀。急性感染的水牛分别于第10 , 13 , 16 和22 周宰杀。肝脏的组织病理学显示, 肝片吸虫幼虫可引起肝脏一系列炎症反应。急性感染后第10 周和13 周肝细胞变性、坏死, 肝小叶内淤血, 大量淋巴细胞浸润, 继而发展为肝索萎缩。急性感染后第22 周及慢性感染后第26 周, 汇管区内有大量结缔组织及新生的小胆管增生。实验表明慢性感染和急性感染肝片吸虫的水牛均可导致肝脏典型的病理学变化。     

Humoral immune response in calves to single-dose, trickle and challenge infections with Fasciola hepatica

In cattle experimentally infected with Fasciola hepatica, parasite specific IgG1 and IgG2 responses were studied. Additionally parasite specific IgE production was assessed by the Passive Cutaneous Anaphylaxis reaction. The primary infection was administered either as a single-dose or as a trickle infection over a 4-week period. Animals were challenged 4 months later. Titres of IgG1 and IgG2 against excretory-secretory parasite products (FhESAg), and against a whole-worm extract (FhSomAg) were measured by enzyme-linked immunosorbent assay (ELISA) in relation to weight gain, serum hepatic enzyme levels, and fluke infection rate. At necropsy, the mean number of flukes recovered was similar in both infected groups. The two ELISAs specific for bovine IgG1 showed analogous sensitivity and specificity (92% and 94%). Cross-reactivity was observed towards Echinococcus granulosus, Cysticercus tenuicollis, and C. ovis but not towards C. bovis, Cooperia spp., and Ostertagia spp. FhESAg gave rise to apparently more stable specific IgG1 titres as compared to FhSomAg. Mean IgG1 titres were significantly higher in the single-dose-infected group than in the trickle-infected group during the early migratory phase of the infection (week 2 to week 4 (FhSomAg) or week 6 (FhESAg)). IgG2 values were consistently lower than IgG1 levels. The kinetic response of both isotypes yielded a similar pattern. Specific IgE antibodies were detected in cattle of both infected groups from week 2 post-primary infection (PPI) onwards. The mean serum glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gammaGT) activities were significantly higher in the single-dose-infected group for 3 weeks around peak levels (12-14 weeks PPI and 14-16 weeks PPI for GLDH and gammaGT respectively). Western blotting revealed a major antigenic fraction in FhESAg (26-30 kDa) recognized specifically by sera from F. hepatica infected calves as early as 6-8 weeks PPI. Experimental challenge caused no statistically significant modification of any parameter (IgG1 and IgG2 titres, enzymatic activities, immunoblotting) used to monitor the course of the infection. No correlation was found between fluke size and number, and antibody titres, suggesting that IgG1 production has little protective effect against F. hepatica infection.     

Worm recovery, haemagglutinating antibodies and IgE-levels after immunisation against Fasciola hepatica in rats

Female inbred Hooded Lister (HL) rats were each infected with 20 metacercariae (Mc) of Fasciola hepatica. Remarkable variations between the number of flukes established in the bile ducts suggest the presence of individual, perhaps genetically controlled, differences in immune responsiveness of HL rats to F. hepatica. Serum (4 ml) from HL rats infected with 20 Mc 6 weeks prior to transfer partially protected rats against a F. hepatica challenge infection. However, 1 X 10(6) lymphoid cells originating from rats of the same age and stage of infection did not show the same protective qualities. Furthermore, attempts to immunise HL rats i.p. with either juvenile or adult excretory/secretory (ES) products, or somatic tissue antigens and AlOH3-gel as adjuvant failed. When compared to other investigations, the present results further suggest that both the adjuvant and the route of administration are crucial for the stimulation of a protective immunity to F. hepatica. Low titers and low anamnestic responses of haemagglutinating antibodies after prior immunisation with juvenile ES antigens or both juvenile ES and somatic tissue antigen suggest the occurrence of an immunosuppressive effect caused by juvenile ES products. The total serum IgE-levels in immunised groups were generally lower when compared to the challenge control group, whereas the F. hepatica ES-specific IgE-levels rose after challenge, but immediately decreased again when compared to challenge controls. These findings support the hypothesis of an immunomodulatory effect caused by the vaccination scheme.     

Immunization of rats against Fasciola hepatica using crude antigens conjugated with Freund's adjuvant or oligodeoxynucleotides

Chronic Fasciola hepatica infection is correlated with the development of a T helper (Th2)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN) or Freund's complete adjuvant (FCA), known to promote a Th1 (T helper 1) immune responses, could provide protection from F. hepatica infection, total homogenate (TH) of F. hepatica mixed with CpG-ODN or FCA were injected subcutaneously (s.c.) into Wistar rats. A F. hepatica-specific Th1-predominant immune response was induced with CpG-ODN or FCA in lymph nodes of immunized animals. Lymph node cells from TH-CpG-ODN or TH-FCA immunized rats showed increased antigen-specific proliferation with high levels of INFgamma, compared to lymphocytes from rats injected with TH alone. In contrast, these two groups of immunized animals did not modify IL-4 release by draining lymph node cells, when they were subsequently stimulated with TH in vitro. However, a significant reduction in the burden of flukes (76.7%) was only observed in rats immunized with TH-FCA. Conversely, immunization of rats with TH-CpG-ODN did not promote protection against the parasite. Therefore, even though CpG-ODNs and FCA induced Th1 type responses, only FCA provided a significant protection to rats infected with F. hepatica.     

Phenotype of hepatic infiltrates and hepatic lymph nodes of lambs primarily and challenge infected with Fasciola hepatica, with and without triclabendazole treatment

The phenotype of the hepatic inflammatory infiltrate and hepatic lymph nodes (HLN) was analysed in lambs primarily and challenge infected with Fasciola hepatica. Group 1 was primarily and challenge infected with two doses of 200 metacercariae (mc) each and was non-treated. Trickle infection was administered to five groups: group 2 was challenge infected and non-treated; group 3 was primarily infected and non-treated; group 4 was primarily infected and treated with triclabendazole (TCBZ) at 12 weeks postinfection (wpi); group 5 was treated at 4 wpi and challenge infected and group 6 was treated at 12 wpi and challenge infected. An uninfected group was used as the control. The distribution of T cell subpopulations (CD3, CD4 and CD8), and B cells (CD79alpha, IgM, IgG) was analysed. The hepatic inflammatory infiltrate was represented mainly by CD3 and CD4 T cells, and B cells (CD79alpha, IgG). These infiltrates were more severe (P < 0.05) in primarily (group 3) or challenge (groups 2, 5 and 6) trickle infected lambs than in the group single challenge infected (group 1). Cellular changes in HLN consisted in an increase of CD4 over CD8 T cells and an increase of B cells and IgG+ plasma cells, and they were more severe in primarily and challenge trickle infected groups than in the group infected with two larger doses of mc, although significant differences were not found with respect to all challenge trickle infected groups. The strong local cellular and humoral immune responses did not protect against subsequent infections, neither in non-treated lambs (group 2) nor in lambs treated with TCBZ at 4 wpi (group 5) or 12 wpi (group 6).