Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Fasciola hepatica primoinfections and reinfections in sheep drive distinct Th1/Th2/Treg immune responses in liver and hepatic lymph node at early and late stages.

The expression of proinflammatory (IL-1ß, IFN-γ, TNF-α) and regulatory (IL-10, TGF-ß, IL-4) cytokines, as well as the transcription factor FoxP3, was quantified in the liver and hepatic lymph node (HLN) of sheep primoinfected and reinfected with Fasciola hepatica at early (4, 8 and 16 days post-infection [dpi]) and late (100 dpi) stages. The liver exerted a Th2 immune response at very early stages after the primoinfection with F. hepatica that induced the downregulation of IFN-γ, followed by a Th1/Th2/Treg response although the late stages were characterised by the expression of Th1/Th2 immune mediators. Contrarily, in reinfected sheep a robust mixed Th1/Th2/Treg immune response was found at very early stages meanwhile at late stages we observed a Th2/Treg immune response overcoming the expression of Th1 immune mediators. However, the HLN displayed a completely different Th1/Th2/Treg expression profile compared to the liver. Primoinfections with F. hepatica in HLN induced a mixed Th1/Th2/Treg environment from early stages, establishing a Th2 immune response at a late stage. However, the reinfected sheep exerted a Th2 immune response at early stages led by the IL-4 expression in opposition to the Th1/Th2/Treg found in the liver, meanwhile at late stages the HLN of reinfected sheep exerted a mixed Th1/Th2/Treg immune response. This is the first work publishing the expression of immune mediators in the liver and HLN from reinfected sheep with F. hepatica. The study of the immune responses exerted by the natural host in the target organs directly implied in the development of F. hepatica are crucial to better understand the immunopathogenesis of the fasciolosis being a key factor to develop effective vaccines.

Efficacy of a multivalent vaccine against Fasciola hepatica infection in sheep.

In this work we report the protection found in a vaccination trial performed in sheep with two different vaccines composed each one by a cocktail of antigens (rCL1, rPrx, rHDM and rLAP) formulated in two different adjuvants (Montanide ISA 61 VG (G1) and Alhydrogel®(G2)). The parameters of protection tested were fluke burden, faecal egg count and evaluation of hepatic lesions. In vaccinated group 1 we found a significant decrease in fluke burden in comparison to both unimmunised and infected control group (37.2%; p = 0.002) and to vaccinated group 2 (Alhydrogel®) (27.08%; p = 0.016). The lower fluke burden found in G1 was accompanied by a decrease in egg output of 28.71% in comparison with the infected control group. Additionally, gross hepatic lesions found in vaccine 1 group showed a significant decrease (p = 0.03) in comparison with unimmunised-infected group. The serological study showed the highest level for both IgG1 and IgG2 in animals from group 1. All these data support the hypothesis of protection found in vaccine 1 group.

Antigen-specific response of CD4+ T cells and hepatic lymph node cells to Fasciola hepatica-derived molecules at the early and late stage of the infection in sheep.

The immunomodulatory capacity of F. hepatica antigens is probably one of the main reasons for the development of a driven non-protective Th2 immune response. In this study, we analysed the cellular response of hepatic lymph node cells and CD4+ T cells in terms of proliferative response, efficiency of antigen presentation and cytokine production, to F. hepatica-derived molecules, at early and late stages of the infection. Thirty-one sheep were allocated into five groups and were slaughtered at 16 dpi and 23 wpi. In order to analyse antigen-specific response, the following F. hepatica recombinant molecules were used: rFhCL1, rFhCL2, rFhCL3, rFhCB1, rFhCB2, rFhCB3, rFhStf-1, rFhStf-2, rFhStf-3 and rFhKT1. A cell proliferation assay using hepatic lymph node cells and an antigen presentation cell assay using CD4+ T cells were performed. At 16 dpi, all molecules but rFhStf-2 and rFhKT1 elicited a significant cell proliferative response on hepatic lymph node cells of infected animals. At both early and late stage of the infection, antigen presentation of rFhCB3 and rFhCL2 resulted in higher stimulation index of CD4+ T cells which was IL-2 mediated, although no statistically significant when compared to uninfected animals. Significant cytokine production (IL-4, IL-10 and IFN-γ) was conditioned by the antigen-specific cell stimulation. No CD4+ T cell exhaustion was detected in infected sheep at the chronic stage of the infection. This study addressed antigen-specific response to F. hepatica-derived molecules that are involved in key aspects of the parasite survival within the host.

Seroprevalence of Toxoplasma gondii and associated risk factors in domestic pigs raised from Cuba.

A cross-sectional study was carried out to determine the seroprevalence of Toxoplasma gondii and associated risk factors in pigs in the largest pork-producing region in Cuba. Serum samples from 420 pigs, including 210 sows and 210 post-weaning pigs, were tested for antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay. Anti-T. gondii antibodies were detected in 56 animals (13.3%, 95% CI: 10.1-16.6). A generalized estimating equations model revealed that the risk factors associated with higher seropositivity in pigs were altitude (higher in farm's location < 250 m above sea level (masl) versus ≥ 250 masl) and age (higher in sows compared to post-weaning pigs). The results indicated that this protozoan parasite is widely distributed on pig farms in the study area, which is a public health concern since the consumption of raw or undercooked pork meat products containing tissue cysts is considered one of the main routes of T. gondii transmission worldwide. Control measures should be implemented to reduce the risk of exposure to T. gondii in pigs in Cuba.

Characterization of dendritic cells and follicular dendritic cells in the hepatic lymph nodes and liver of sheep experimentally infected with Fasciola hepatica.

Fasciola hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the hepatic lymph nodes (HLN) and livers of sheep during the early stages of infection with F. hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. hepatica.

Fasciola hepatica induces Foxp3 T cell, proinflammatory and regulatory cytokine overexpression in liver from infected sheep during early stages of infection.

The expression of T regulatory cells (Foxp3), regulatory (interleukin [IL]-10 and transforming growth factor beta [TGF-ß]) and proinflammatory (tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1ß) cytokines was quantified using real time polymerase chain reaction (qRT-PCR) in the liver of sheep during early stages of infection with Fasciola hepatica (1, 3, 9, and 18 days post-infection [dpi]). Portal fibrosis was also evaluated by Masson's trichrome stain as well as the number of Foxp3+ cells by immunohistochemistry. Animals were divided into three groups: (a) group 1 was immunized with recombinant cathepsin L1 from F. hepatica (FhCL1) in Montanide adjuvant and infected; (b) group 2 was uniquely infected with F. hepatica; and (c) group 3 was the control group, unimmunized and uninfected. An overexpression of regulatory cytokines of groups 1 and 2 was found in all time points tested in comparison with group 3, particularly at 18 dpi. A significant increase of the number of Foxp3+ lymphocytes in groups 1 and 2 was found at 9 and 18 dpi relative to group 3. A progressive increase in portal fibrosis was found in groups 1 and 2 in comparison with group 3. In this regard, group 1 showed smaller areas of fibrosis than group 2. There was a significant positive correlation between Foxp3 and IL-10 expression (by immunohistochemistry and qRT-PCR) just as between portal fibrosis and TGF-ß gene expression. The expression of proinflammatory cytokines increased gradually during the experience. These findings suggest the induction of a regulatory phenotype by the parasite that would allow its survival at early stages of the disease when it is more vulnerable.

Revealing the Prevalence of Toxoplasma in Sierra Morena's Wild Boar: An ELISA-Based Study Using Meat Juice.

This research work focused on the prevalence of Toxoplasma gondii in wild boar from the Sierra Morena region. We conducted an ELISA analysis using meat juice samples. A total of 892 samples from six hunting seasons (2013-2019) were collected from the provinces that constitute the Sierra Morena Mountain range. These samples were analyzed using the Pigtype® ELISA kit, specifically developed for detecting T. gondii in meat juice. The overall prevalence of T. gondii in Sierra Morena was 23.2%. The highest prevalences were observed in Córdoba (31.6%) and Jaén (25.9%). These provinces exhibit the highest density of wild boar as well as the greatest presence of the Iberian lynx (Lynx pardinus). Further in-depth studies are necessary, but it appears that the presence of wild felids and scavenger behavior may be associated with this observation.

Serosurveillance of Trichinella sp. in wild boar and Iberian domestic suids in Mediterranean ecosystems of southwestern Spain.

AIMS: A cross-sectional study was carried out to assess the seroprevalence and risk factors associated with Trichinella spp. exposure in wild boar and Iberian domestic pigs from Mediterranean ecosystems of southwestern Spain. METHODS AND RESULTS: Serum samples from 1360 wild boar and 439 Iberian domestic pigs were obtained during 2015-2020, from regions where Iberian pigs are raised under extensive conditions, hence sharing habitat with wild boar. Seropositivity was found in 7.4% (100/1360; 95% CI: 6.1-8.9) of the wild boar analysed. In this species, the individual seroprevalence ranged from 3.6% (8/223) (hunting season 2016-2017) to 11.4% (37/326) (2018-2019). A significant higher seropositivity was observed during the hunting season 2018-2019 (p < 0.009: OR = 3.07; 95% CI = 1.32-7.18) and one statistically significant cluster was detected within the studied area, in south central Andalusia [Relative Risk (RR) = 2.9; p = 0.037]. Females showed a significantly higher seroprevalence than males (8.7% vs. 5.8%) (p < 0.001: OR = 1.58; 95% CI = 1.08-2.32). No seropositivity to Trichinella spp. was detected in Iberian domestic pigs (0.0%; 95% CI: 0.0-0.9). CONCLUSIONS: Although wild boar play an important role as a reservoir of Trichinella sp. in the Mediterranean ecosystems of southwestern Spain, our results suggest that the wild boar production system does not seem to pose a risk of Trichinella exposure to domestic pigs, despite sharing habitats in these ecosystems.

Liver Histopathological and Immunohistochemical Evaluation from Fasciola hepatica Experimentally Infected and Reinfected Sheep.

Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since the current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed to design effective vaccines. To date, few studies have evaluated host-parasite interaction by comparing infected and reinfected animals. The present study evaluates the microscopical hepatic lesions in sheep infected and reinfected with Fasciola hepatica during the acute and chronic stages of infection. The histopathological study revealed the presence of necrotizing foci (NF1) associated with larvae migration during the early stages of infection in the primoinfected (PI) and reinfected (RI) groups. In the late stages of infection of the PI group and at the early and late stages of infection in the RI groups, extensive necrotizing/hemorrhagic foci (NF2) were found in the vicinity of enlarged bile ducts, some containing adult flukes, suggesting parasites may have caused NF2 while feeding. The immunohistochemical study revealed an increase in Foxp3+ T cells in both PI and RI groups with respect to the UC group and in the infiltrates adjacent to NF1 in the RI groups with respect to the PI group, suggesting the F. hepatica induce Foxp3 T cell expansion to facilitate parasite survival. In addition, in both the PI and RI groups, and during acute and chronic stages of the infection, a poor expression of iNOS was found accompanied by a strong expression of CD163, suggesting a marked M2 activation of macrophages in the hepatic lesions, which may be related with healing processes, and it also may facilitate parasite survival. The main differences between PI and RI animals were the more severe infiltration of eosinophils and Foxp3+ T cells, whereas RI did not modify M2 activation of macrophages which occurs since the early stages of primoinfection.

Seroprevalence and genetic characterization of Toxoplasma gondii in domestic pigs intended for human consumption in Cuba.

Domestic pigs are considered as one of the main intermediate hosts in the zoonotic transmission of Toxoplasma gondii in many countries. Serological and molecular studies are warranted to better understand the epidemiology and transmission patterns of this parasite worldwide. To date, seroepidemiological information on T. gondii in domestic pigs in Cuba is very scarce and there are no reports of T. gondii genotypes circulating in this country. Here, we aimed to estimate the seroprevalence of T. gondii and provide genetic characterization of the strains circulating in slaughtered pigs intended for human consumption in Central Cuba. Seroprevalence was determined in 450 serum samples from slaughtered pigs in Villa Clara province using ELISA. Anti-Toxoplasma gondii IgG antibodies were detected in 100 animals (22.2%, 95% CI: 18.5-26.2). Conventional PCR of the 529-bp marker of T. gondii was performed in hearts and diaphragm tissues of all ELISA-seropositive pigs. Toxoplasma gondii DNA was detected in four animals. Further genetic characterization of the positive DNA samples was performed by multilocus PCR-RFLP and PCR-sequencing typing tools. Molecular analysis revealed four different genetic profiles that were combinations of type I, II, III and u-1 alleles, suggesting the circulation of non-clonal genotypes of T. gondii in domestic pigs in Cuba. Our results indicate that T. gondii is widely distributed in slaughtered pigs in this country, which might have important implications for public health. To the best of our knowledge, this is the first report on genetic characterization of T. gondii in Cuba. Although preliminary, the results suggest a high genetic diversity of T. gondii in the study region. Further studies based on parasite isolation are needed to definitively identify the genotypes circulating and characterize the virulence of strains detected in pigs in Cuba, and to assess the risk of zoonotic transmission from pork products in this country.

Fasciolosis: pathogenesis, host-parasite interactions, and implication in vaccine development.

Fasciola hepatica is distributed worldwide, causing substantial economic losses in the animal husbandry industry. Human fasciolosis is an emerging zoonosis in Andean America, Asia, and Africa. The control of the disease, both in humans and animals, is based on using anthelmintic drugs, which has resulted in increased resistance to the most effective anthelmintics, such as triclabendazole, in many countries. This, together with the concerns about drug residues in food and the environment, has increased the interest in preventive measures such as a vaccine to help control the disease in endemic areas. Despite important efforts over the past two decades and the work carried out with numerous vaccine candidates, none of them has demonstrated consistent and reproducible protection in target species. This is at least in part due to the high immunomodulation capacity of the parasite, making ineffective the host response in susceptible species such as ruminants. It is widely accepted that a deeper knowledge of the host-parasite interactions is needed for a more rational design of vaccine candidates. In recent years, the use of emerging technologies has notably increased the amount of data about these interactions. In the present study, current knowledge of host-parasite interactions and their implication in Fasciola hepatica vaccine development is reviewed.

Study of the migration of Fasciola hepatica juveniles across the intestinal barrier of the host by quantitative proteomics in an ex vivo model.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

Transcriptomic Analysis of Ovine Hepatic Lymph Node Following Fasciola hepatica Infection - Inhibition of NK Cell and IgE-Mediated Signaling.

Fasciola hepatica is a trematode parasite responsible for major economic losses in livestock production, and is also a food-borne zoonotic agent in developing rural regions. For years, the immunoregulatory mechanisms employed by the parasite have hampered efforts to develop a successful vaccine candidate. Given that a comprehensive understanding of the immune response to infection is needed, we investigated the gene expression changes in ovine hepatic lymph nodes after experimental infection with F. hepatica. Lymph nodes from uninfected and infected animals were processed for RNA sequencing (RNA-seq) at 16 weeks post-infection. Comparison of groups revealed 5,132 differentially-expressed genes (DEGs). An inhibition of pro-inflammatory pathways, which has previously been described during fasciolosis, was evident in our data. However, other signals previously identified in ruminant peripheral blood mononuclear cells (PBMC) or liver tissue, such as activation of TGF-ß or apoptosis-related pathways were not detected. We found inhibition of some key immunological pathways, including natural killer (NK) cell activity and IgE-mediated signaling. These may point to additional some as yet unrecognized mechanisms employed by the parasite to evade the host immune response. Understanding these, and leveraging information from this and other omics studies, will be important for the development of future vaccine prototypes against this parasite.

Timing of Transcriptomic Peripheral Blood Mononuclear Cell Responses of Sheep to Fasciola hepatica Infection Differs From Those of Cattle, Reflecting Different Disease Phenotypes.

Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in sheep can have more severe effects, potentially leading to death. Previous transcriptomic analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell activation, and inhibition of nitric oxide production in macrophages in response to infection. However, the differences between ovine and bovine responses have not yet been explored. The objective of this study was to further investigate the transcriptomic response of ovine peripheral blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences between ovine and bovine PBMC responses. Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially expressed (DE) genes between infected and control animals at each time point (analysis 1), and for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study performed previously on bovine PBMC. A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor signalling, which suggested that an activation of innate response to intracellular nucleic acids and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the previous bovine transcriptomic study revealed that anti-inflammatory response pathways which were significantly overrepresented in the acute phase in cattle, including IL-10 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, as compared with sheep, may be related to the general absence of acute clinical signs in cattle. These findings offer scope for "smart vaccination" strategies for this important livestock parasite.

Potential Influence of Helminth Molecules on COVID-19 Pathology.

In recent months, the parasitology research community has been tasked with investigation of the influence of parasite coinfection on coronavirus disease 2019 (COVID-19) outcomes. Herein, we share our approach to analyze the effect of the trematode Fasciola hepatica as a modulator of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and of COVID-19 pathology.

A Partially Protective Vaccine for Fasciola hepatica Induced Degeneration of Adult Flukes Associated to a Severe Granulomatous Reaction in Sheep.

Fasciolosis is an important economic disease of livestock. There is a global interest in the development of protective vaccines since current anthelmintic therapy is no longer sustainable. A better knowledge of the host-parasite interaction is needed for the design of effective vaccines. The present study evaluates the microscopical hepatic lesions in sheep immunized with a partially protective vaccine (VAC1), a non-protective vaccine (VAC2), and an infected control group (IC). The nature of granulomatous inflammation associated with degeneration of adult flukes found in the VAC1 group was characterized by immunohistochemistry. Hepatic lesions (fibrous perihepatitis, chronic tracts, bile duct hyperplasia, infiltration of eosinophils and lymphocytes and plasma cells) were significantly less severe in the VAC1 group than in the IC group. Dead adult flukes within bile ducts were observed only in the VAC1 group and were surrounded by a severe granulomatous inflammation composed by macrophages and multinucleate giant cells with a high expression of lysozyme, CD163 and S100 markers, and a low expression of CD68. Numerous CD3+ T lymphocytes and scarce infiltrate of FoxP3+ Treg and CD208+ dendritic cells were present. This is the first report describing degenerated flukes associated to a severe granulomatous inflammation in bile ducts in a F. hepatica vaccine trial.

Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics.

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen.

Protection against Fasciola hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of hepatic enzymes, faecal egg count and hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and hepatic lymph nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross hepatic lesions of 56.6% (p < 0.001) and reduced hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.

Identification of protective peptides of Fasciola hepatica-derived cathepsin L1 (FhCL1) in vaccinated sheep by a linear B-cell epitope mapping approach.

BACKGROUND: Fasciolosis is one of the most important parasitic diseases of livestock. The need for better control strategies gave rise to the identification of various vaccine candidates. The recombinant form of a member of the cysteine protease family, cathepsin L1 of Fasciola hepatica (FhCL1) has been a vaccine target for the past few decades since it has been shown to behave as an immunodominant antigen. However, when FhCL1 was used as vaccine, it has been observed to elicit significant protection in some trials, whereas no protection was provided in others. METHODS: In order to improve vaccine development strategy, we conducted a linear B-cell epitope mapping of FhCL1 in sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus Montanide and with significant reduction of the fluke burden, sheep vaccinated with FhCL1, FhHDM, FhLAP and FhPrx plus aluminium hydroxide and with non-significant reduction of the fluke burden, and in unvaccinated-infected sheep. RESULTS: Our study showed that the pattern and dynamic of peptide recognition varied noticeably between both vaccinated groups, and that the regions 55-63 and 77-84, which are within the propeptide, and regions 102-114 and 265-273 of FhCL1 were specifically recognised only by vaccinated sheep with significant reduction of the fluke burden. In addition, these animals also showed significant production of specific IgG2, whereas none was observed in vaccinated-Aluminium hydroxide and in infected control animals. CONCLUSIONS: We have identified 42 residues of FhCL1 that contributed to protective immunity against infection with F. hepatica in sheep. Our results provide indications in relation to key aspects of the immune response. Given the variable outcomes of vaccination trials conducted in ruminants to date, this study adds new insights to improve strategies of vaccine development.