Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Humoral and cellular immune responses in rats during a primary infestation with Fasciola hepatica.

The antibody and lymphocyte responses to Fasciola hepatica were studied in rats. Infested rats were shown to produce antibodies against excretory-secretory (ES) products of adult flukes as early as the first week after infestation. Immunoblotting revealed fractions of ES products of adult flukes to which antibodies were progressively produced during the course of the infestation. Proliferation of peripheral blood lymphocytes, splenocytes and thymocytes when incubated with different mitogens (Concanavalin A (ConA) or Pokeweed mitogen (PWM) or different liver fluke antigens (metacercariae antigen (EM) or ES products of adult flukes) have been studied. In response to these mitogens or antigens, splenocytes were stimulated on the second and fourth weeks after infestation. Thymocytes were significantly activated by PWM on the second week but peripheral blood lymphocytes did not show any statistically significant response. Results obtained in antibody production, immunoblotting and lymphocyte proliferation suggested sequential releases of F. hepatica substances and the existence of common proteins between adult and juvenile parasite stages. Cellular and humoral responses observed in this work did not seem to confer a complete resistance to liver fluke primary infestation on the rat.     

Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system

The dot-ELISA reaction was used to study the dynamics of IgG titers in cattle naturally infected with Fasciola hepatica after anthelmintic treatment and during reinfection. Excretion/secretion products (ES) of the parasite were used as antigens for the dot-ELISA. IgG antibodies were no longer detectable by dot-ELISA, 4-6 months after nine animals received the first of three weekly doses of triclabendazole (15 mg kg(-1)) and were then maintained on a pasture free of F. hepatica metacercariae. Six fluke-free cattle began shedding F. hepatica eggs 3-6 months after grazing a pasture contaminated with metacercariae of the parasite. A detectable increase in dot-ELISA IgG antibody levels was observed 2-4 weeks after natural reinfection by grazing a similar pasture contaminated with F. hepatica metacercariae. The usefulness of the dot-ELISA system to diagnose chronic infection by serology is complicated by previous treatment against the parasite. It is concluded that the ES antigens can be useful to detect early infection of cattle with F. hepatica in a dot-ELISA system     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos)

Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.     

comparison of an indirect haemagglutination assay and an Elisa for diagnosing Fasciola hepatica in experimentally and naturally infected sheep

Summary

An enzyme‐linked immunosorbent assay (ELISA) with somatic (S) or excretory‐secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono‐infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.     

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

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

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.     

Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fag) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fag and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.     

Immune responses in rats and cattle to primary infections with Fasciola hepatica

Antibody and lymphocyte proliferative responses to fluke antigens were compared in rats and cattle following infection with Fasciola hepatica. Antibody responses differed between rats and cattle in that rats responded more quickly and with a greater rate of synthesis over the first three weeks of infection than did cattle. Lymphocyte proliferative responses developed and disappeared at similar times in both cattle and rats, being detectable early in infection, but returning to background levels by week 6 after infection. The presence of antibody but not of lymphocyte proliferative responses correlated with the timing of the development of resistance in cattle and rats as described by others. Sensitisation by intraperitoneal injection of fluke antigens in Freund's incomplete adjuvant (FIA) induced antibody production but not lymphocyte proliferative responses in both rats and cattle. Serum antibodies continued to rise after oral infection of sensitised animals although this was much more marked in the cattle than in the rats. On the other hand lymphocyte proliferative responses were absent when sensitised cattle were infected, while the findings with rats were much more variable. Cattle sensitised by intraperitoneal injection of fluke antigens in FIA were not protected against subsequent infection with F hepatica.     

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

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

Partial purification of somatic and excretory-secretory products of adult Fasciola hepatica and their application for the serodiagnosis of experimental and natural fascioliasis using an ELISA

Six partially purified antigen fractions from adult Fasciola hepatica (three somatic tissues [Fhs] and three excretory products [Fhm]) were used in a micro-ELISA to monitor the serum antibody levels of an experimental rabbit F hepatica infection. Fhs 1 detected infection after 19 to 26 days and the titre remained significantly higher than that of the controls until day 103 of infection (end of experiment). Using Fhm 1 and Fhm 2, antibodies were detected between 12 and 19 days after infection. Fhm 2 distinguished infected from uninfected rabbits during the entire experimental period, whereas Fhm 1 did not. Excretory-secretory products of a low molecular weight were also antigenic and could differentiate between infected animals and controls. One hundred and nine sera from naturally infected cattle and uninfected controls were tested with the same antigens. Although antibody was detected, the results were inconsistent and further purification of the antigens may eventually improve the sensitivity of the method.     

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

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

Acquired immunity to Fasciola hepatica in splenectomised rats.

Significant resistance to a second infection with Fasciola hepatica was obtained in splenectomised and sham operated rats (P less than 0.001, 78 and 76 per cent respectively) when compared with singly infected splenectomised or sham operated control groups. For both the stimulating and challenge infections, there were no significant differences in the number of flukes recovered from rats that had been splenectomised as compared to those receiving the sham operation. Thus, splenectomy did not significantly affect the ability of rats to develop an acquired immunity to F hepatica, nor were fluke recoveries from a particular schedule of infection significantly affected by the presence of absence of a spleen. It is concluded that the presence of a spleen is not necessary for the development of acquired immunity to F hepatica in the rat.     

Attempts to immunize rats against infection with Fasciola hepatica using in vitro culture antigens from newly excysted metacercariae.

Attempts were made to immunise rats against Fasciola hepatica using the culture products obtained from the in vitro cultivation of newly excysted metacercariae. Three culture regimes were chosen: (1) medium NCTC 135 for 48 h (2) NCTC 135 + 20 per cent fetal calf serum (FCS) for 48 h (3) NCTC 135 + 20 per cent FCS for 14 days. The used culture medium from each of these regimes was concentrated, mixed with adjuvant and injected subcutaneously into rats. Similarly treated unused culture media was used in control rats. The rats were challenged with an oral dose of 20 F hepatica metacercariae 35 days later and autopsied 96 days after the start of the experiment. The fluke burdens in those rats which had received the culture antigens did not differ significantly from those in the control groups.     

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

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

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

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

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

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

Identification and isolation of a specific antigen with diagnostic potential from Dictyocaulus viviparus.

The purpose of the investigation was to isolate and identify a specific antigen of Dictyocaulus viviparus that can be used to diagnose lungworm infections in cattle. Somatic, excretion and secretion antigens of adult D. viviparus and somatic antigens of L3 larvae were examined in an indirect enzyme-linked immunosorbent assay (ELISA) to determine whether they cross-reacted with sera collected from calves with mono-infections of Fasciola hepatica. Ostertagia ostertagi, Ascaris suum, or Cooperia oncophora. Serum samples containing antibodies directed against F. hepatica, A. suum, and O. ostertagi cross-reacted with somatic antigens of adult D. viviparus; these sera cross-reacted less with excretion and secretion antigens. When somatic antigens of adult D. viviparus were analysed in a Western blot, a 17-kDa protein that did not react with the heterologous sera was detected. This protein was isolated by ultrafiltration and anion chromatography. Sera collected from calves infected with D. viviparus was tested in indirect ELISAs with either somatic antigens of adult D. viviparus or with a low molecular antigen fraction of this preparation containing the 17-kDa protein. The extinction values that were measured in both assays correlated well. We conclude that the 17-kDa protein isolated from somatic antigens of adult D. viviparus may be useful in developing an improved immunoassay to diagnose lungworm infections in cattle.