Immunization with the recombinant myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant elicits a Th1-biased immune response and a reduction of parasite burden in Fasciola hepatica infected rats

The aim of this study was to assess the immune response and the protective efficacy elicited by the vaccination with the recombinant Fasciola hepatica myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant against the infection with F. hepatica in rats. Four groups of 15 animals each were used for the study, one group was immunized with the recombinant F. hepatica MRLC in Adjuplex® adjuvant and the other groups remained as adjuvant, positive and negative control groups. The parasitological study showed that a statistically significant reduction of 65.1% and 82.1% in fluke burden and fecal egg count, respectively, was detected in vaccinated animals. In addition, vaccination with FhrMRLC induced a well-defined humoral and cellular immune response characterized by a significant production of specific IgG and IL-2, IL-12, TNF-α and IFN-γ; which confirms the immunogenic capacity of the FhrMRLC.     

Vaccination with cathepsin L phage-exposed mimotopes,single or in combination,reduce size,fluke burden,egg production and viability in sheep experimentally infected with Fasciola hepatica

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1 × 10¹³ filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1 + CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16 weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1 + CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1 + CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P < 0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

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

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

Lymphocyte responses of rats vaccinated with cDNA encoding a phosphoglycerate kinase of Fasciola hepatica (FhPGK) and F. hepatica infection

Lymphocyte responses in the blood, peritoneal fluid and both mesenteric and hepatic lymph nodes of cDNA-FhPGK/pCMV vaccinated and/or Fasciola hepatica infected rats of both sexes were investigated to provide an insight into the immune responses that develop in different body compartments. The immune response that developed in cDNA-FhPGK/pCMV vaccinated females contributed to partial protection against F. hepatica infection (54% reduction in fluke recovery), while more liver flukes were found in the livers and bile ducts of cDNA-FhPGK/pCMV vaccinated male rats than in unvaccinated animals (increase of 13%). Rat sex not only affected the ultimate effectiveness of vaccination but also lymphocyte responses following vaccination and/or infection. Different CD4 + and CD8 + T cell profiles were noted in peritoneal fluid and lymph nodes, but not in blood, during acute and chronic fasciolosis. Moreover, independent lymphocyte responses developed in distinct body compartments. Immune responses of rats were polarized towards Th2/Treg with lymphocytes isolated from male rats showing higher IL-4 and IL-10 production than females. Lymphocyte proliferative capacities in response to mitogen (PHA) or vaccine antigen (FhPGK) were impaired in both sexes with a considerably higher reduction observed for males and restored lymphocyte proliferative capacities reported for females vaccinated with cDNA-FhPGK/pCMV during chronic fasciolosis.     

Resistance to experimental infection with Fasciola hepatica in exotic and domestic breeds of sheep

Significant breed differences were detected in fecal egg counts and fluke counts following experimental infection of five breeds of sheep with metacercariae of F. hepatica. Two experiments compared the responses of Barbados Blackbelly, St. Croix, Florida Native, Finn X Rambouillet and Targhee sheep to primary and challenge infections (Expt. 1) and to multiple infections (Expt. 2). Barbados Blackbelly sheep were more susceptible to infection than the other breeds in both experiments. St. Croix and Florida Native sheep were the most resistant breeds in Expt. 1, while Targhee and Florida Native were the most resistant in Expt. 2. There was no evidence of acquired immunity in any breed in either experiment.     

Capillaria hepatica: Relation of structure and composition of egg shell to antigen release

Clean, nonembryonated Capillaria hepatica eggs recovered from infected liver tissue by physical methods as well as eggs obtained after passage through the mouse gastrointestinal tract were examined with the electron microscope. In eggs collected by physical methods the outer membrane showed defects where it was lacking at various points or disrupted. The pillars of the outer shell were frequently broken or fractured resulting in virtual collapse or separation of the outer shell from the inner shell. No shell matrix was observed in any eggs examined. Even more dramatic effects were observed in eggs recovered following passage through the mouse gastrointestinal tract. Clean, nonembryonated eggs collected by physical methods were suspended at 5 C in phosphate-buffered saline. A large amount of protein was released initially into the medium; the amount released then fell to a low level at which it remained for several weeks. Gel diffusion tests with concentrated protein supernatant and C. hepatica egg-derived antigen were compared using appropriate antisera. Bands of identity were present in both; however, egg antigen contained other proteins not present in egg supernatant. These studies indicate that during physical collection of C. hepatica eggs, sufficient damage occurs to allow for the release of materials into host tissue during experimental egg granuloma formation. An hypothesis is presented concerning the viability of C. hepatica eggs in host liver tissue following host cellular response and the possible modes of action which trigger development of eggs after release from infected liver.     

Fasciola hepatica: Comparison of the sedimentation and FLOTAC techniques for the detection and quantification of faecal egg counts in rats

We compared the sedimentation and FLOTAC techniques for the detection and quantification of Fasciola hepatica eggs in faecal samples obtained from 120 experimentally-infected rats before intervention, and in 42 rats after drug administration. Additionally, the average time for a single test was determined. A single FLOTAC showed a higher sensitivity (92.6%) than 2, 4 and 8 sedimentation readings (63.0-85.2%) for detecting F. hepatica eggs in rat faeces post-treatment. On average, it took 21 min to prepare and examine a single FLOTAC, whereas 114 min were needed for the sedimentation method including the reading of 8 slides. In both treated and untreated rats, the sedimentation method resulted in higher mean faecal egg counts (FECs) than FLOTAC (P < 0.05). In view of the high sensitivity and efficiency, the FLOTAC technique holds promise for experimental work in the F. hepatica-rat model. Additional research is needed to determine the reasons for the observed differences in FECs.     

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.     

Immunity acquired by sheep from an experimental infection with haemonchus contortus

Adams D.B. and Beh K.J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology11: 381–386. A primary infection of sheep with a single dose of Haemonchus contortus larvae was traced by faecal egg counts until it had substantially declined after 55 weeks. These primed sheep were then given a sequence of two reinfections with the parasite. Comparison of faecal egg counts in primed sheep and in two separate groups of previously worm-free sheep showed that primary infection conferred significant immunity. This, however, was not sufficiently protective to prevent the development of further anaemia and faecal egg counts indicative of clinical haemonchosis. It is suggested that an adaptation in the host-parasite relationship which promotes the longevity of primary infection with H. contortus may also moderate the induction of acquired immunity.The titre of haemagglutinating antibody specific for H. contortus rose in serum during the course of primary infection, but the two reinfections did not stimulate a rise in titre. Titres of haemagglutinating antibody before reinfection did not correlate with subsequent faecal egg counts.     

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.     

Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica

Background

Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology.

Methodology

A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21).

Results

We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death.

Conclusion

The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis.     

Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro

Sandeman R. M. and Howell M. J. 1982. Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro. International Journal for Parasitology12: 467–471. The role of sheep antibodies which precipitate with surface antigens of Fasciola hepatica is unclear. In an attempt to clarify their function these antibodies were characterized as to their immunoglobulin class and ability to affect the survival of fluke in rats. The ability of fluke antigens complexed with sheep antibody to vaccinate rats against infection was also tested. IgM antibodies were involved in precipitate formation on the teguments of fluke 3 weeks after infection but IgG₁ predominated at later stages of infection. The decreased survival of fluke in rats after culture with increasing levels of sheep antibodies suggests that the antibodies exert some deleterious effect on the fluke in vitro. The fluke antigen-sheep antibody complex failed to immunize rats against infection. Since sheep antibodies to F. hepatica can impair the ability of fluke to resist further attack in rats but not sheep, it is suggested that some effector mechanism other than antibody is defective in the latter.     

Evaluation of losses in carcasses of cattle naturally infected with Fasciola hepatica: effects on weight by age range and on carcass quality parameters

Although fasciolosis is a relatively common disease, the productive and economic losses resulting from cattle with chronic fasciolosis are unclear. This paper aims to investigate the effect of fasciolosis on the parameters of carcass quality and discuss the hypothesis that the effects on weight differ among age ranges of cattle. For this, we analysed abattoir data of 30,151 bovines, from 928 farms, slaughtered in Uruguay in 2016, of which 33.9% (95% confidence interval (CI): 27.3–41.1%) had Fasciola hepatica (liver fluke). A mixed model was built to assess whether the effect of fasciolosis on weight differs depending on the age range, using the interaction term ‘age*F. hepatica’. The effect on the carcass parameters was tested using a proportional logistic regression. The interaction of age and F. hepatica was statistically significant (P < 0.001). Differences in carcass weights between infected and non-infected animals were observed mostly at younger ages (up to 30 months), with the highest difference observed in the 23–30 months age range (estimated marginal mean difference of 6.34 kg). Overall, the presence of F. hepatica was positively associated with poor conformations and lower fat scores of carcasses (P < 0.001). The carcasses of cattle infected with F. hepatica had 0.16 times greater odds of having worse conformation scores than carcasses of cattle without F. hepatica (proportional odds ratio (POR) = 1.16; 95% CI: 1.07–1.26). Similarly, carcasses of cattle with F. hepatica had 0.30 times (POR = 1.30, 95% CI: 1.23–1.39) greater odds of having poorer fat scores than carcasses of cattle without F. hepatica. Therefore, infection with F. hepatica is associated with poorer carcass quality parameters and lower weights, and the effect on weight differs across age ranges.     

The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

Dineen, J. K. and Windon, R. G. (1980). The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology10: 189–196. Rams selected for responsiveness and unresponsiveness to vaccination with irradiated T. colubriformis larvae at an early age were mated to unselected random bred ewes. Progeny were vaccinated with 20,000 irradiated larvae at 8 and 12 weeks of age, given anthelmintic treatment at 16 weeks and challenged with 20,000 normai larvae at 17 weeks. The results, based on wether worm counts and ewe faecal egg counts, showed significant differences between responder and non-responder progeny. There was a significant correlation between worm counts and faecal egg counts of half-sibs from the same sire group. The occurrence of globule leucocytes was inversely related to worm burdens of wether progeny, however, no clear relationship was found with eosinophils. In vitro lymphocyte stimulation using T. colubriformis L₃ antigen, concanavalin A and lipopolysaccharide showed that statistically defined responder progeny, pooled from both responder and non-responder sire groups, gave higher responses than non-responder lambs after vaccination. The results confirm that genetically-determined factors are involved in the response of lambs to vaccination at an early age, and indicate that rapid genetic progress may be achieved in the type of mating usually carried out under fleld conditions.     

Investigations of 'transfer factor' activity in immunity to Ostertagia circumcincta and Trichostrongylus colubriformis infections in sheep.

Immunity was successfully transferred by ‘Transfer Factor’ prepared from leucocytes of two adult Scottish Blackface donor rams infected with O. circumcincta and T. colubriformis to 4-month-old susceptible Fin X Dorset lambs. The immunity was expressed by a significantly reduced faecal egg count and worm burden compared to challenged, untreated controls. The immunity was comparable to that produced in another group of lambs given an initial infection prior to challenge with both parasites.     

Liver fluke vaccines in ruminants: strategies,progress and future opportunities

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30–54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.     

Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase.

Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M(r) ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M(r) range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti-Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5-10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.