Evaluation of Reproductive Histology Response of Adult Fasciola hepatica in Goats Vaccinated with Cathepsin L Phage-Exposed Mimotopes.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis' gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

Diagnosis of fasciolosis antibodies in Brazilian cattle through ELISA employing both native and recombinant antigens.

Bovine fasciolosis is a parasitic disease with a global reach. Coprological based on egg detection in fecal samples and liver inspection to evaluate the presence of the parasite is currently the gold standard for diagnosing chronic fasciolosis in cattle. However, these techniques are labor-intensive and ineffective during the acute phase of the disease. Serodiagnosis using native and recombinant antigens has become an interesting alternative in efforts to identify cattle fasciolosis. We evaluated cattle from abattoir (n = 139) and farms (n = 500) through liver inspection and coprological examination, respectively. Our laboratory team optimized and validated enzyme-linked immunosorbent assay tests based on somatic antigen, excretory/secretory proteins, and the recombinant antigen cathepsin L-1 to detect serum antibodies against fasciolosis in cattle. For animals from abattoir, 10 were positive for fasciolosis according to liver inspection. Both FhES and FhrCL-1 presented an area under the receiver operating characteristic (AUROC) curve of 0.80, with a sensitivity of 0.80 (95% CI: 0.46-0.95) and 0.70 (95% CI: 0.38-0.90) and specificity of 0.81 (95% CI: 0.73-0.87) and 0.87 (95% CI: 0.80-0.92), respectively. For those cattle from farms, 28 were positive only for fasciolosis according to coprological examination. In this scenario, FhES gave the best performance, with an AUROC of 0.84, sensitivity of 0.79 (95% CI: 0.60-0.90), and specificity of 0.86 (95% CI: 0.82-0.89). In conclusion, our study highlights the potential of serodiagnosis for accurately screening cattle fasciolosis. The promising sensitivity and specificity values of FhES when compared to liver inspection or coprological examination enhance its importance for cattle fasciolosis diagnosis. IMPORTANCE: The aim of this article was to identify antibodies against fasciolosis in cattle in Brazil. The methodology was reproduced in our laboratory and applied for the first time to the Brazilian cattle herd. The antigens tested can be used as a screening test and thus speed up the diagnosis of bovine fascioliasis.

Plasma cytokines during acute human fascioliasis.

Fascioliasis is a foodborne trematode endemic worldwide. Children under 15 years have the highest prevalence of infection. We hypothesized that acute fascioliasis would be associated with more pronounced cytokine changes than in chronic disease or no helminth infections. To test this hypothesis, 33 children who lived in the Peruvian highlands were classified into 3 groups: acute fascioliasis, chronic fascioliasis, and no helminth infection. Type Th1, Th2, and Th17 cytokines were measured in plasma by cytometric bead array. Children with acute infection had higher levels of IL-5 and IL-17 compared with controls (p < 0.001 and p < 0.007, respectively). The increased IL-5 plasma concentration in children with acute infection was associated with the eosinophilia found in that group.

Stunting in pre-school and school-age children in the Peruvian highlands and its association with Fasciola infection and demographic factors.

Fascioliasis is a zoonotic trematode infection that is endemic in the highlands of Peru. Chronic fascioliasis can be asymptomatic and remain undiagnosed for years. Chronic malnutrition in children, as manifested by stunting, leads to delayed cognitive development and lost productivity. We hypothesized that fascioliasis is among the factors associated with stunting in children from endemic areas. We conducted a cross-sectional study among children attending pre-school and school in 26 communities in the Anta province in the Cusco region of Peru. We conducted interviews to collect information on demographic, socioeconomic, and medical history. Blood was collected and tested for complete cell count and FAS2 ELISA for Fasciola antibodies. Three stool samples per participant were tested for parasites by Kato-Katz and Lumbreras rapid sedimentation methods. Chronic fascioliasis was determined by the presence of ova in stool. Children's height, weight, and age were recorded and used to calculate height for age Z scores (HAZ). Three thousand children participated in the study. Nine percent (264) of children had at least one positive test for Fasciola infection, 6% (164) had chronic fascioliasis, and 3% (102) had only positive antibody tests. The median HAZ was -1.41 (IQR: -2.03 to -0.81) and was similar in males and females. Twenty six percent (776) of children had stunting with HAZ < -2. Children with chronic fascioliasis had a lower median HAZ than children without Fasciola (-1.54 vs. -1.4, p = 0.014). History of treatment for malnutrition, history of treatment for anemia, having other helminths in stool, lower socioeconomic score, living at a higher elevation, and fewer years of schooling of both parents were associated with a lower HAZ score. In a multiple regression analysis, older age and a lower socioeconomic score were associated with a lower HAZ score. While fascioliasis and other helminths were associated with lower HAZ, they were not independent of the socioeconomic score.

Fasciola hepatica co-infection enhances Th1 immune response in the adventitial layer of non-fertile Echinococcus granulosus cysts.

Cystic echinococcosis is a widespread zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. In intermediary hosts, two types of echinococcal cysts can be found: fertile, which produce protoscoleces, the infective form of the parasite to dogs; and infertile, that do not present protoscoleces and therefore are not able to continue with the parasite life cycle. The adventitial layer, the local immune response against the cyst, plays an important role in cyst fertility. Grazing cattle can often feature Fasciola hepatica co-infection, a parasite known to modulate the host systemic immune response. In this work the cellular Th1/Th2 immune profiles were evaluated in the adventitial layer of fertile and non-fertile cysts with and without co-infection with Fasciola hepatica. Measuring with immunohistochemistry and qPCR in adventitial layer, we report that non-fertile cysts present higher levels of Th1 cytokines (IFN-γ (P < 0.0001) and TNF-α (P < 0.05)), and fertile cysts have higher levels of Th2 cytokines (IL-4 (P < 0.001)). Co-infection with Fasciola hepatica is associated with a decrease in the expression of IL-4 (P < 0.05) and an increase in the expression of IFN-γ (P < 0.0001) in the adventitial layer of fertile cysts. Non-fertile cysts were associated with higher levels of Th1 cytokines in the adventitial layer, with IFN-γ expression enhanced by F. hepatica co-infection (P < 0.0001), confirming that polyparasitism should be considered in the treatment and control of naturally infected cattle.

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

Eosinophils Control Liver Damage by Modulating Immune Responses Against Fasciola hepatica.

Eosinophils are granulocytes that participate in the defense against helminth parasites and in hypersensitivity reactions. More recently, eosinophils were shown to have other immunomodulatory functions, such as tissue reparation, metabolism regulation, and suppression of Th1 and Th17 immune responses. In the context of parasitic helminth infections, eosinophils have a controversial role, as they can be beneficial or detrimental for the host. In this work, we investigate the role of eosinophils in an experimental infection in mice with the trematode parasite Fasciola hepatica, which causes substantial economical losses around the world due to the infection of livestock. We demonstrate that eosinophils are recruited to the peritoneal cavity and liver from F. hepatica-infected mice and this recruitment is associated with increased levels of CCL11, TSLP, and IL-5. Moreover, the characterization of peritoneal and hepatic eosinophils from F. hepatica-infected mice showed that they express distinctive molecules of activation and cell migration. Depletion of eosinophils with an anti-Siglec-F antibody provoked more severe clinical signs and increased liver damage than control animals which were accompanied by an increase in the production of IL-10 by hepatic and splenic CD4+ T cells. In addition, we also report that eosinophils participate in the modulation of humoral immune responses during F. hepatica infection, contributing to their degranulation. In conclusion, we demonstrate that eosinophils are beneficial for the host during F. hepatica infection, by limiting the production of IL-10 by specific CD4+ T cells and favoring eosinophil degranulation induced by specific antibodies. This work contributes to a better understanding of the role of eosinophils in parasitic helminth infections.

Cattle co-infection of Echinococcus granulosus and Fasciola hepatica results in a different systemic cytokine profile than single parasite infection.

E. granulosus is a cestode that causes Cystic Echinococcosis (CE), a zoonotic disease with worldwide presence. The immune response generated by the host against the metacestode induces a permissive Th2 response, as opposed to pro-inflammatory Th1 response. In this view, mixed Th2 and regulatory responses allow parasite survival. Overall, larval Echinococcus infections induce strong regulatory responses. Fasciola hepatica, another common helminth parasite, represents a major infection in cattle. Co-infection with different parasite species in the same host, polyparasitism, is a common occurrence involving E. granulosus and F. hepatica in cattle. 'While it is known that infection with F. hepatica also triggers a polarized Th2/Treg immune response, little is reported regarding effects on the systemic immune response of this example of polyparasitism. F. hepatica also triggers immune responses polarized to the Th2/ Treg spectrum. Serum samples from 107 animals were analyzed, and were divided according to their infection status and Echinococcal cysts fertility. Cytokines were measured utilizing a Milliplex Magnetic Bead Panel to detect IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-18. Cattle infected only with F. hepatica had the highest concentration of every cytokine analyzed, with both 4.24 and 3.34-fold increases in IL-10 and IL-4, respectively, compared to control animals, followed by E. granulosus and F. hepatica co-infected animals with two-fold increase in IL-10 and IL-4, compared to control animals, suggesting that E. granulosus co-infection dampens the cattle Th2/Treg immune response against F. hepatica. When considering Echinococcal cyst fertility and systemic cytokine concentrations, fertile cysts had higher IFN-γ, IL-6 and IL-18 concentrations, while infertile cysts had higher IL-10 concentrations. These results show that E. granulosus co-infection lowers Th1 and Th2 cytokine serological concentration when compared to F. hepatica infection alone. E. granulosus infections show no difference in IFN-γ, IL-1, IL-2, IL-6 and IL-18 levels compared with control animals, highlighting the immune evasion mechanisms of this cestode.

Fasciola hepatica coinfection modifies the morphological and immunological features of Echinococcus granulosus cysts in cattle.

Polyparasitism occurs when animals harbour multiple parasites concomitantly. It is a common occurrence but is generally understudied in wild and domestic animals. Fasciola hepatica and Echinococcus granulosus, which are helminths of ungulates, frequently coinfect cattle. The effects of this particular type of polyparasitism are not well documented. The metacestode of Echinococcus granulosus is surrounded by the adventitial layer, which constitutes the host immune response to the parasite. This layer in cattle is produced by a granulomatous reaction and is involved in echinococcal cyst (EC) fertility. Due to the systemic immune-modulating abilities of Fasciola hepatica, coinfection possibly generates a favourable environment for EC growth. A total of 203 Echinococcus granulosus sensu stricto cysts were found in 82 cattle, of which 42 ECs were found in 31 animals coinfected with Fasciola hepatica. The overall infection intensity was 3 cysts per animal. Coinfection with Fasciola hepatica decreased the mean infection intensity to 1.4 cysts per animal. Regarding EC size, coinfection resulted in smaller ECs (15.91 vs 22.09 mm), especially for infertile lung cysts. The adventitial layer of ECs in coinfected animals lacked lymphoid follicles and palisading macrophages, which are generally hallmarks of the granulomatous immune response. The ECs in coinfected animals had organized laminated layers, whereas those in animals without coinfection did not. Although coinfection was not statistically associated with EC fertility, we did not find fertile cysts in the livers of coinfected animals. We concluded that coinfection with Fasciola hepatica and Echinococcus granulosus has a detrimental effect on ECs, particularly infertile cysts.

Study of Eosinophil Apoptosis Induced by Fasciola hepatica Excretory-Secretory Products.

The excretory-secretory products released by the liver fluke Fasciola hepatica (FhESP) are in close contact with the immune system and have different immunomodulatory effects associated with the parasite virulence. The control of the early immune response is crucial for the establishment of the fluke in the host. Related to this, eosinophils (Eo) are implicated as effector cells in helminthic infections, and the induction of Eo apoptosis has been demonstrated to be a remarkable immunoevasion mechanism induced by the parasite. In this chapter, we describe different techniques to assay Eo apoptosis triggered by FhESP as well as the mechanisms involved in this phenomenon.

Evaluation of the Immune Regulatory Properties of Dendritic Cells During Fasciola hepatica Infection.

Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells, initiating the adaptive immune response. Ex vivo DC cultures are useful to evaluate how helminths regulate DC maturation and stimulatory activity. Here, we describe how to isolate CD11c+ from F. hepatica-infected mice to evaluate their activation state, cytokine production and regulatory function in an allogeneic T cell assay.

Liver Fluke Vaccine Assessment in Cattle.

Liver fluke Fasciola hepatica remains an important agent of foodborne trematode disease producing great economic losses due to its negative effect on productivity of grazing livestock in temperate areas. The prevailing control strategies based on antihelminthic drugs are not long term sustainable due to widespread resistance. Hence, vaccination appears as an attractive option to pursue for parasite eradication.

Design of a Peptide-Carrier Vaccine Based on the Highly Immunogenic Fasciola hepatica Leucine Aminopeptidase.

Many studies have shown that the degree of organization and repetitiveness of an antigen correlates with its efficiency to induce a B-cell response and production of neutralizing antibodies. Here we describe the design of a chimeric protein based on the hexamer form of the highly immunogenic Fasciola hepatica leucine aminopeptidase as a carrier system of small peptides with potential use as a multiepitope vaccine.

Bulk tank milk ELISA to detect IgG1 prevalence and clustering to determine spatial distribution and risk factors of Fasciola hepatica-infected herds in Mexico.

Fasciola hepatica is a helminth parasite that causes huge economic losses to the livestock industry worldwide. Fasciolosis is an emerging foodborne zoonotic disease that affects both humans and grazing animals. This study investigated the associations between climatic/environmental factors (derived from satellite data) and management factors affecting the spatial distribution of this liver fluke in cattle herds across different climate zones in three Mexican states. A bulk-tank milk (BTM) IgG1 enzyme-linked immunosorbent assay (ELISA) test was used to detect F. hepatica infection levels of 717 cattle herds between January and April 2015. Management data were collected from the farms by questionnaire. The parasite's overall herd prevalence and mean optical density ratio (ODR) were 62.76% and 0.67, respectively. The presence of clustered F. hepatica infections was studied using the spatial scan statistic. Three marked clusters in the spatial distribution of the parasite were observed. Logistic regression was used to test three models of potential statistical association from the ELISA results using climatic, environmental and management variables. The final model based on climatic/environmental and management variables included the following factors: rainfall, elevation, proportion of grazed grass in the diet, contact with other herds, herd size, parasite control use and education level as significant predictors. Geostatistical kriging was applied to generate a risk map for the presence of parasites in dairy herds in Mexico. In conclusion, the spatial distribution of F. hepatica in Mexican cattle herds is influenced by multifactorial effects and should be considered in developing regionally adapted control measures.

Anticuerpos de cadena única de alpaca para la detección de antígenos de Fasciola hepática / Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens

RESUMEN Objetivo. Producir anticuerpos recombinantes de cadena única de alpaca que se unan con alta afinidad y especificidad al antígeno excretado-secretado (ES) de Fasciola hepatica para el desarrollo de tecnologías nuevas de diagnóstico de fascioliasis humana y animal. Materiales y métodos. Se ha construido una genoteca de cADNde los dominios variables de anticuerpos de cadena única pesada, conocidos como VHH, a partir de células mononucleares de sangre periférica de una alpaca inmunizada con el antígeno ES de F. hepatica. La genoteca fue tamizada con el antígeno ES por despliegue diferencial de fagos (phage display), seleccionando diez VHH que se unen específicamente a ES. El VHH anti ES fue clonado en un vector de expresión, la proteína recombinante (VHH-ES1) de 15,3 kDa fue producida por fermentación en E. coli y purificada a homogeneidad por cromatografía de afinidad. La unión del VHH-ES1 al antígeno ES fue evaluada por ELISA usando VHH-ES1 como anticuerpo de captura, antisuero policlonal anti-ES de conejo y conjugado anti IgG de conejo con peróxidasa de rábano. Resultados. Se ha identificado y producido un VHH-ES1 recombinante que se une al antígeno ES (VHH-ES1) que correspondía a un anticuerpo de la subclase IgG2 de bisagra larga. La unión del anticuerpo VHH-ES1 al antígeno muestra linealidad respecto a la concentración de ES en el rango de 50-5000 ng/mL y el valor límite de detección del antígeno está en el rango de 30-170 ng/mL de ES (R2=0,99). Conclusión . El VHH-ES1 se une con afinidad y especificidad al antígeno ES de F. hepatica y es un anticuerpo promisorio a evaluar para el desarrollo de nuevas tecnologías de diagnóstico de fascioliasis.

ABSTRACT Objectives. To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. Materials and Methods. A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. Results. A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). Conclusions. The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

Risk factors for the presence of Fasciola hepatica antibodies in bulk-milk samples and their association with milk production decreases, in Cuban dairy cattle.

BACKGROUND: Worldwide, Fasciola hepatica infection causes high production losses in the livestock industry. Recently, studies have analyzed the association between measurements of F. hepatica infection intensity and herd management practices. The aim of the present study, the first of its kind in a subtropical region, was to evaluate associations between F. hepatica bulk-tank milk ELISA results with herd management factors and milk yield in dairy herds, in Camagüey, Cuba. The SVANOVIR® F. hepatica-AB ELISA was used to measure F. hepatica antibody levels in a random sample of 516 dairy herds during the period of May-July of 2014. Farm management practice data were collected using a questionnaire. RESULTS: With 82% of the herds testing positive, the results indicate that F. hepatica is very widespread in this area. Reductions in milk production of 18 and 32% were observed in herds with Optical Density Ratios (ODR) of 0.3-0.6 and > 0.6, respectively, when compared to herds with ODR <  0.3. Overall, the longer the milking cows were put out to pasture, the higher the levels of anti-parasite antibodies. Co-grazing with sheep and goats also significantly increased the risk of high ODR. CONCLUSIONS: Our data show a widespread occurrence of the parasite as well as a major potential impact of the infection on the Cuban development goal of becoming self-sufficient in milk production. Our risk factor analysis suggests that the prevention of infection around water sources, and the separation of cattle from small ruminants could be useful control measures. This is the first epidemiological survey of F. hepatica abundance, and associated reductions in milk yield, in dairy herds in Cuba.

Socioeconomic Factors Associated with Fasciola hepatica Infection Among Children from 26 Communities of the Cusco Region of Peru.

Fasciola hepatica is the most widely distributed trematode-affecting humans. The Andes Mountains are highly endemic for fascioliasis. We report results of a cross-sectional study evaluating the epidemiology of Fasciola among children in 26 agricultural communities in the Cusco region of Peru. Children 3 to 16 years old were enrolled in preschools and schools. Blood from participants was tested for complete blood counts, transaminases, and Fasciola antibodies. Stool samples were tested for Fasciola and other parasites. A total of 2,515 children were included in the analysis and the mean age was 9.6 years (±3.6). Ten percent (253) of the children had at least one positive test for Fasciola, 6% had chronic infection, and 0.4% acute infection. The rest of the subjects had only antibodies against Fasciola. The prevalence of infection varied from 0% to 20% between communities. Children with evidence of Fasciola exposure were older, lived at higher altitudes, and had a lower socioeconomic status than children without infection. The logistic regression analysis showed that children from Ancahuasi district, older children, and children with higher measures of poverty were more likely to have Fasciola exposure. Fascioliasis is common in the Cusco region and associated with poverty. However, the distribution varies markedly between communities.

Low allelic diversity in vaccine candidates genes from different locations sustain hope for Fasciola hepatica immunization.

Fasciola hepatica is a trematode parasite that causes fasciolosis in animals and humans. Fasciolosis is usually treated with triclabendazole, although drug-resistant parasites have been described in several geographical locations. An alternative to drug treatment would be the use of a vaccine, although vaccination studies that have been performed mainly in ruminants over the last 30 years, show high variability in the achieved protection and are not yet ready for commercialisation. Since F. hepatica exhibits a high degree of genomic polymorphism, variation in vaccine efficacy could be attributed, at least partially, to phenotypic differences in vaccine candidate sequences amongst parasites used in the challenge infections. To begin to address this issue, a collection of F. hepatica isolates from geographically dispersed regions, as well as parasites obtained from vaccination trials performed against a field isolate from Uruguay and the experimentally maintained South Gloucester isolate (Ridgeway Research, UK), were compiled to establish a F. hepatica Biobank. These collected isolates were used for the genetic analysis of several vaccine candidates that are important in host-parasite interactions and are the focus of the H2020 PARAGONE vaccine project (https://www.paragoneh2020.eu/), namely FhCL1, FhCL2, FhPrx, FhLAP and FhHDM. Our results show that F. hepatica exhibits a high level of conservation in the sequences encoding each of these proteins. The consequential low variability in these vaccine candidates amongst parasites from different geographical regions reinforces the idea that they would be suitable immunogens against liver fluke isolates worldwide.

[Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens]. / Anticuerpos de cadena única de alpaca para la detección de antígenos de Fasciola hepática.

OBJECTIVES.: To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. MATERIALS AND METHODS.: A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. RESULTS.: A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). CONCLUSIONS.: The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

OBJETIVO.: Producir anticuerpos recombinantes de cadena única de alpaca que se unan con alta afinidad y especificidad al antígeno excretado-secretado (ES) de Fasciola hepatica para el desarrollo de tecnologías nuevas de diagnóstico de fascioliasis humana y animal. MATERIALES Y MÉTODOS.: Se ha construido una genoteca de cADNde los dominios variables de anticuerpos de cadena única pesada, conocidos como VHH, a partir de células mononucleares de sangre periférica de una alpaca inmunizada con el antígeno ES de F. hepatica. La genoteca fue tamizada con el antígeno ES por despliegue diferencial de fagos (phage display), seleccionando diez VHH que se unen específicamente a ES. El VHH anti ES fue clonado en un vector de expresión, la proteína recombinante (VHH-ES1) de 15,3 kDa fue producida por fermentación en E. coli y purificada a homogeneidad por cromatografía de afinidad. La unión del VHH-ES1 al antígeno ES fue evaluada por ELISA usando VHH-ES1 como anticuerpo de captura, antisuero policlonal anti-ES de conejo y conjugado anti IgG de conejo con peróxidasa de rábano. RESULTADOS.: Se ha identificado y producido un VHH-ES1 recombinante que se une al antígeno ES (VHH-ES1) que correspondía a un anticuerpo de la subclase IgG2 de bisagra larga. La unión del anticuerpo VHH-ES1 al antígeno muestra linealidad respecto a la concentración de ES en el rango de 50-5000 ng/mL y el valor límite de detección del antígeno está en el rango de 30-170 ng/mL de ES (R2=0,99). CONCLUSIÓN: El VHH-ES1 se une con afinidad y especificidad al antígeno ES de F. hepatica y es un anticuerpo promisorio a evaluar para el desarrollo de nuevas tecnologías de diagnóstico de fascioliasis.

Anti-Fas2 IgM antibodies in Fasciola hepatica infected patients with positive IgG serology.

Background: Fascioliasis is an infectious disease caused by parasites Fasciola hepatica and F. gigantica. Humans are infected by the consumption of vegetables and water contaminated with the infective form of the parasite. Materials and Methods: In this study, an IgM-ELISA with the cysteine proteinase Fas2 antigen was evaluated with sera from 76 patients infected with F. hepatica, 24 patients with other parasite infections and 84 healthy volunteers. Results: IgM-ELISA resulted in 43% positives in F. hepatica patients with positive serology to Fas2-ELISA, but no positives resulted from testing healthy volunteers and individuals infected with other parasites. The IgM-ELISA diagnostic parameters showed a sensitivity of 43.4% (95% CI 0.321-0.553), specificity of 100% (95% CI 0.957-1), and no cross-reactivity with other parasitic infection. Interference by rheumatoid factor in the IgM immunoassay was controlled by treating sera with rheumatoid factor absorbent before testing. Conclusions: Fas2 antigen is detected by circulating IgM in patients infected with F. hepatica and IgM-ELISA using Fas2 appears as a specific immunoassay to detect the acute phase of the acute phase of F. hepatica infection in humans.