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.

Protective Role of Purified Cysteine Proteinases against Fasciola gigantica Infection in Experimental Animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-gamma, and TNF-alpha, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-beta, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

Experimental Murine Fascioliasis Derives Early Immune Suppression with Increased Levels of TGF-beta and IL-4

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19+ B cells was observed as early as week 1 post-infection while CD4+/CD8+ T cells were down-regulated. Accumulation of Mac1+ cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-alpha mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1beta expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-beta were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-beta and IL-4 during the early stages of infection.

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 percent (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.

Evaluation of the stability of coated plates with antigen at different temperatures and times by ELISA test to diagnose fasciolosis

Considering that ELISA method presently is the test of choice for diagnosis of fasciolosis, the present study was undertaken to evaluate the maximum validity of coated plates at different temperatures and different times during one year of evaluation. Serum samples of patients infected with fasciolosis [n=10], hydatidosis [n=5], toxocariasis [n=5], and negative control sera [n=5] were examined. Two series of plates were considered. The first series were coated with Fasciola homogenate Ag 12 micro g/ml, and after some steps were blocked with gelatin and preserved at different temperatures as -80 °C, -20 °C, -4 °C and +4°C. The 2[nd] series were treated under the same criteria but were not blocked with gelatin. Each series were examined by ELISA test from 1[st] month to 12[th] month. Sera with 1:125 dilution, and peroxidase-conjugated goat anti-human IgG diluted 1:10000 were considered optimum. To ease reporting the results and due to many similarities only results related to 1[st], 6[th] and 12[th] months were analyzed and sensitivity, specificity plus cut-off were determined for each series separately. Preserving the coated plates, while unblocked at -80°C for 6-8 months is pertinent and functional and in that case, we can be sure the best out put would be applicable

Evaluation of purified tegumental antigen of Fasciola adult worm In diagnosis of human fasciollash

This study was designed to develop a sandwich ELISA for detection of Fasciola antigens in stool and sera of fascioliasis patients as a better diagnostic alternative to routine parasitological methods. Anti-Fasciola antibodies were produced by immunization of rabbit with Fasciola purified tegumental antigen [PTA] obtained from worms collected from livers of cattle infected with Fasciola. Raised antibodies were then employed in sandwich ELISA for detection of Fasciola antigen in collected sera and stool samples. In this study sera and stool samples from 40 Fasciola infected patients, 30 patients infected with other parasites [Schistosoma, Hydatid and Ancklystoma] and 20 uninfected individuals were tested by sandwich ELISA for detection of Fasciola antigen. The sensitivity of coproantigen assay reached 90% for detection of Fasciola antigens in stool and 87.5% for detection of Fasciola antigen in sera of fascioliasis patients. The specificity of the assay was 94% for stool samples and 92% for sera of negative controls and patients harboring other parasites collectively. The diagnostic efficacy of the assay was 92.2% and 90% for stool and serum samples, respectively. Moreover, a positive correlation was found between ova count in stool of Fasciola infected patients and antigen levels in both sera and stool samples [r= 0.456, p< 0.01; r=0.532, p< 0.01]. In conclusion, our data demonstrated that the employment of rabbit anti-Fasciola IgG antibodies in sandwich ELISA for the detection of Fasciola coproantigen in stool provided a sensitive and specific tool for immunodiagnosis of Fasciola infection

Seroprevalence of fasciolosis and the difference of fasciolosis between rural area and city center in Isparta, Turkey

To investigate the seroprevalence of fasciolosis and the possible causes of differences between rural and city center. We undertook a multi-stage sampling analysis of data from Isparta, Turkey, between March and June 2004. Four hundred and fifteen individuals participants from Isparta center and 171 from Asagi Gokdere village were included in the study. Fasciola hepatica [F. hepatica] specific antibodies were analyzed using excretory-secretory [ES]-enzyme-linked immunosorbent assay [ELISA] method. Fasciola hepatica antibodies were detected as positive in 10 [2.4%] of 415 people whose sera were collected from the city center and 16 [9.3%] of 171 people from Asagi Gokdere village. The positivity rates between village and city center were found statistically significant. A statistical difference was noted for fasciolosis positivity between individuals who have ingested water cress and who have not. Fasciolosis was not detected in the individuals who used to wash vegetables with water containing vinegar. Most of the patients in this region reported consumption of uncooked or unwashed water cress. Watering channel is one of the major risk factors of fasciolosis. Therefore, it is essential to determine the watering systems in this region. Moreover, ES-ELISA would be useful in investigating the laboratory diagnosis of fasciolosis

Serum interferon-gamma and interleukins-6 and -8 during infection with Fasciola gigantica in cattle and buffaloes

This study investigated the presence of cytokines interferon (IFN)-gamma, interleukins (IL) -6 and -8 in serum of cattle and buffaloes infected with Fasciola gigantica from one to 16 weeks post-infection to determine their T cell response during infection. The concentration of these cytokines was determined by sandwich enzyme-linked immunosorbent assay (ELISA). No IFN-gamma was detected in these animals while IL-6 was elevated from one to 16 weeks postinfection. Levels of IL-8 were also elevated in infected buffaloes from one to 16 weeks post-infection. A predominantly T helper (Th) 2 response which started early in the infection was apparently present in cattle and buffaloes in this study which was characterised by IL-6. IL-8 production could be another mechanism of immune response in buffaloes during infection with F. gigantica.

Fasciola gigantica: Immunization of rabbits with proteins isolated from copropantigen

Two fractions were isolated from coproantigen by ion-exchange chromatography, in which DEAE cellulose was utilized. Both fractions and crude antigen were characterized by SDS-polyacrylamide gel electrophoresis, which revealed 13 bands of molecular weight ranged from 205-31 in crude coproantigen. While, fraction I was resolved into 6 bands of molecular weights of 198, 178, 148, 111, 101 and 45. Fraction II showed seven bands of 191 kDa, 178 kDa, 166 kDa, 118 kDa, 98.5 kDa, 72 kDa and 32 kDa. Fraction II showed higher immunoreactivity than fraction I by ELISA. Three immunoreactive bands of 191 kDa, 118 kDa and 98.5 kDa were identified in fraction II using immunoblot assay. Five bands of 178 kDa, 148 kDa, 111 kDa, 101 kDa and 45 kDa were detected in fraction I. Immunization of rabbits twice with fraction II in Freund's adjuvant with two-week interval, followed by challenge with F. gigantica metacercariae resulted in 66.6% protection from infection. The protection was assessed by the detection of hepatic damage, worm recoveries and antibody response. A high level of IgG response in vaccinated rabbits than control infected ones occurred and was found to be responsible for the recorded protection

Biliary fascioliasis: Magnitude of the problem and role of immunodiagnosis

This study was carried out to evaluate the prevalence of fascioliasis, which is a relatively common, but usually underestimated problem in many rural areas in Egypt and to assess its role in causing biliary disorders using the recent technique of cystatin capture ELISA for immunodiagnosis of such cases.Between July 1999 and July 2001, 3218 individuals [2409 males and 809 females] with ages ranged from 6-70 years were selected by stratified random sampling from EI-Husseinya, Sharkeya Governorate, all these individuals were subjected to thorough history taking, clinical examination, stool examination using modified kato-thick smear method, serodiagnosis using cystatin capture ELISA technique. Patients with history suggestive of hepatobiliary disease were subjected to further radiological examination e.g abdominal ultrasonography, C, T abdomen and sometimes ERCP, also routine laboratory investigations were done for this group of patients e.g complete liver function tests and complete blood picture. The overall prevalence of fascioliasis among the present group of patients was 3.7%, the incidence was higher among adolescent [> 12 - 18 years] and adult [> 18 - 40 years] and much less in younger [6-12 years] and older [more than 40 years] age groups. Out of these fasciola positive patients only 10 patients [8.5%] suffered form biliary disorders as proved by abdominal ultrasonography and occasionally ERCP, these constituted about 3.4% of total number of patients who gave history suspecting biliary disorders [N=298]. The cystatin capture ELISA was used to detect anti-fasciola cystatin proteinase antibodies in sera of all fasciola copro positive group [118 patients] and also in a randomly selected group of copro negative patients [358 patients] with relevant or irrelevant signs and symptoms suggesting biliary affection. The ELISA capture test was positive in 110 out of 118 copro positive patients [93.2%] and it was negative in 352 out of 358 copro negative patients. This proved the highly specificity and sensitivity of this test in diagnosis of fascioliasis. The present community based study clarified the magnitude of fascioliasis as a health problem in rural area in Egypt and the significance of it as an aetiological agent of biliary disorders and the value of cystatin capture ELISA in picking up such cases

Análisis preliminar de la respuesta IgM específica en la fascioliasis hepática humana mediante inmunoelectrotransferencia / Preliminary report of the specific IgM response in human hepatic fascioliasis by means of immunoelectrotransfer blot

Enzyme immunoelectrotransfer blot (EITB) has received increasing attention as a confirmative technique for the serodiagnosis of different infectous diseases. Also, EITB has bee used for identifying immunodominant polipeptides as potential antigenic components of parasite's extracts. In this study we used EITB to detect IgM specific antibodies in sera of patients with parasitological confirmed fascioliasis. Our prelininary results show that IgM reactivity was observed in 9 of the 17 sera of infected patients. The sensitivity of EITB was 52.9 percent, with at least 10 IgM immunoreactive bands. Futher studies are needed to define the specificity of the bands observed and to detect IgM immunoreactive polipeptides of Fasciola hepatica in patients with pre-patent fascioliasis. In the parasitological confirmed human infections by F. hepatica the sensitivity of IgM EITB was low and similar to those observed by using IgM ELISA

Inmunodiagnóstico de fasciolosis bovina mediante Elisa y Western Blot / Immunodiagnosis of bovine fasciolosis by Elisa and Western Blot

Se evaluó y caracterizó la respuesta inmune humoral de bovinos naturalmente infectados con fasciola hepatica frente a un extracto total de antígenos de excreción-secreción (E-S) y a una fracción antigénica semipurificada cromatográficamente (<30 kDa), seleccionada previamente por su eficiencia diagnóstica en otras especies. Ambos preparados antigénicos fueron analizados mediante ELISA en microplaca y electroforesis en geles de poliacrilamida en condición de denaturación (SDS-PAGE) y posterior inmunoelectrotransferencia enzimática o Western blot. Para ello se emplearon 52 sueron de bovinos con fasciolosis comprobada mediante examen post mortem, 18 sueros de animales sin la infección y 48 sueros de vacunos infectados con hidatidosis, pero sin fasciolosis. La sensibilidad y especificidad obtenidas en el ELISA con el extracto crudo E-S fueon de 53 por ciento y 100 por ciento, respectivamente, en tanto que con la fraccion semipurificada a igual especificidad (100 por ciento) se presentó una sensibilidad mayor (90 por ciento). A través de Western blot, empleando el extracto antigénico total E-S hubo un reconocimiento específico de bandas correspondientes a los 14,22,27-29 y 37-38 kDa. Las bandas de 37-38 y 27-29 kDa destacaron por su sensibilidad y frecuencia, identificadas por el 90 por ciento y 100 por ciento de los infectados, respectivamente. Con el antígeno semipurificado, se evidenció una banda polipeptídica de 28-30 kDa, reconocida por todos los sueros con fasciolosis. Los resultados demuestran, por un lado, la existencia de diversas fracciones polipeptídicas que potencialmente pueden ser promisorias en el inmunodiagnóstico de fasciolosis bovina

UltramicroELISA indirecto para la detección de anticuerpos IgG en pacientes con fascioliasis / Indirect ultramicroELISA for the detection of IgG antibodies in patients with fascioliasis

Se describe la normalización de un ultramicroELISA para la detección de anticuerpos IgG anti-antígenos de excreción-secreción de Fasciola hepatica (UME-Fasciola). Se estudió un numeroso grupo de sueros entre los cuales 56 eran de pacientes con fascioliasis, 168 eran de pacientes con otras enfermedades parasitarias y 300 procedían de personas sanas, las cuales fueron utilizadas como controles negativos. Respecto al examen parasitológico considerado como "Regla de Oro", el UME-Fasciola mostró una sensibilidad del 100 por ciento, especificidad de 98 por ciento y valores predictivos para positivos y negativos de 90,3 y 100 por ciento, respectivamente. Sólo se observó reacción cruzada con los sueros de pacientes infectados con Opistorchis felineus. Al comparar el UME-Fasciola con el ELISA convencional se obtuvo un índice de concordancia de 95,5 por ciento

Humoral immune response against fasciola hepatica in bovines: primary characterization of protection induced by irradiated metacercariae

There is an increasing awareness of fasciolosis as an important human and veterinary disease. In order to study the humoral inmune response of bovines, 5-month old calves were experimentally inmunized using 1000 ç irradiated metacercariae per animal, and challenged six weeks later with 500 normal ones per calf. Profiles of antibody responses of non inmunized animals following challenge, measured by ELISA, using both excretion/secretion ans somatic antigens, showed a sharp increase in antibody titres in the first 3 weeks post-infection which were maintained throughout the infection. Animals which instead were inmunized with inactivated parasites had similar antibody responses, but the antibody titres were higher. No booster effect was observed when these inmunized animals were challenger. Result of western blot analysis are consistent with these observations: antibodies against high molecular weight parasite-specific bands, ranging from 72,5 to 40 KD, appeared in week 2 PI in inmunized animals, and were mantained throughout infection, importanfly, inmunization resulted in a 84,2 percent reduction in parasite burden, indicating that high circulating antibody titres against these antigens probably are involved in a protective response. The results presented here indicate that, irradiated metacercariae of fasciola hepatica can be used as useful tool to generate a normal antigenic stimulus for the development of circulation antibodies, and toidentify antigens invloved in triggering a protective inmune response

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Identificación y aislamiento de antígenos comunes de Fasciola hepatica / Identification and insolation of common antigens of Fasciola hepatica

Se identificaron por Western blotting y purificaron por cromatrografía de afinidad 4 polipéptidos antigénicos presentes en los antígenos de excreción-secreción que eran comunes a los antígenos somáticos y tegumentarios de Fasciola hepatica empleando para ello el anticuerpo monoclonal ES78. Los pesos moleculares calculados para estos polipéptidos oscilan en el rango de 37 a 13 kd y los mismos demostraron ser altamente reactivos con sueros de animales infectados experimentalmente con Fasciola hepatica en períodos tan tempranos como la segunda semana de infección, con máximos en las semanas 6 y 10

Evaluación de la contrainmunoelectroforesis y la prueba de ELISA en el diagnóstico de la fasiolasis experimental de ratones / Evaluation of the counterelectrophoresis-CEP and the enzyme-linked immunosorbent assay-ELISA in the diagnosis of fasiolasis in mice

Dos pruebas serológicas (contrainmunoelectroforesis-CEP y el ensayo inmunoenzimático-ELISA) fueron utilizadas para hacer un seguimiento longitudinal de 28 ratones infectados con Fasciolasis hepatica. Un grupo de 8 y 10 ratones fueron infectados con 1-2 metacercarias. Los anticuerpos séricos fueron detectados por ELISA a partir de la 1-2da. semana postinfección. Uno de los ratones recién alcanzó un valor de absorbancia diagnóstica a la 5ta. semana. Las precipitinas fueron demostradas por la CEP a la 2-3ra. semana de infección. Sólo en dos casos con diagnóstico comprobado de fasciolasis, no se detectaron precipitinas. Un tercer ratón, positivo mediante ELISA a la 5ta. semana, recién mostró reactividad a la 8va. semana. La prueba de ELISA fue ligeramente más sensible que la CEP para la detección temprana de la infección.

Detección de péptidos inmunorreactivos de Fasciola hepatica, con sueros de personas infectadas, mediante enzimo-inmunoelectrotransferencia / Detection of immunoreactive peptides of Fasciola hepatica, with sera from infected subjects, by immunoelectrotransfer

In order to observe the immunoreactive peptides in a crude extract of adult Fasciola hepatica specimens, proteins were separated by 10// sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and transfered to nitrocellulose paper. Next, sera from 14 human cases of F. hepatica infection, parasitologically confirmed, which presented high titers of specific antivodies aginst F. hepatica detected by ELISA, were reacted with the blotted peptides and immunodetected by an anti-human IgG conjugated with horse-radish peroxidase. SDS-PAGE showed at least 18 bands ranging 96 to 14Kd. Groups of peptides weighting 94-66 Kd, 43-36 Kd and 35-14 Kd reacted with serum antibodies of 12 fascioliasis patients. In the remaining to cases, reactive peptides were not clearly observed. The 94-66 Kd components were immunoreactive with 12 out of the 14 serum samples. On the other hand, 43-36 Kd peptides reacted with 4 of the 14 sera and only 3 out of the 14 sera of infected individuals showed reaction with 30-14 Kd. F. hepatica infection induces in humans diverse antivody responses, being 94-66 Kd bands the most immunoreactive peptides and would be potential serodiagnostic antigens