Immunodiagnosis of current fasciolosis in sheep naturally exposed to Fasciola hepatica by using a 2.9 kDa recombinant protein

A 2.9 kDa recombinant-Fasciola hepatica protein (FhrAPS) was employed to estimate the prevalence of fasciolosis in sheep maintained under field conditions. For this purpose, 340 samples with known status in relation to fasciolosis by using a direct-ELISA and the coprological sedimentation were used. These samples were analysed by using an indirect-ELISA (iELISA) and the FhrAPS recombinant protein and excretory/secretory antigens (FhES) of this trematode. Current fasciolosis (CF) was named when results were positive to antigenemia and/or coprology. Out of 198 sheep with current fasciolosis, 68% were positive to the FhrAPS-ELISA test and 53% to the FhES. We observed 14% of the CF-neg sheep were positive to the FhrAPS, whereas this percentage was 52% with the FhES. A significant correlation between FhrAPS and current fasciolosis was obtained (r2=0.513, p=0.001). We concluded that the FhrAPS provides a more suitable antigen than FhES for developing field trials to know the prevalence of early and current fasciolosis.     

The serum glucose and beta-hydroxybutyrate levels in sheep with experimental Fasciola hepatica and Fasciola gigantica infection

The influence of Fasciola hepatica and Fasciola gigantica infection on serum glucose and beta-hydroxybutyrate (beta-HOB) in sheep was evaluated. This was done by setting up two groups of sheep. The first group (n=13) was split in two sub-groups, one experimentally infected with F. hepatica (n=9) and the other (n=4) as uninfected control. A second group consisting of a sub-group experimentally infected with F. gigantica (n=9) the other sub-group (n=6) left as uninfected control was also set up. The results of weight gain, parasitological and serum liver enzymes activity (glutamate dehydrogenase [GLDH] and gamma glutamyltransferase [gamma-GT]) used in monitoring the infection showed that all infected animals developed fasciolosis. It was observed that a reduction in serum glucose levels was significantly lower (p<0.05) in F. hepatica infected sheep than in uninfected control sheep starting from 5 weeks post-infection (wpi) to the end of the experiment. Similar reduction was recorded in F. gigantica infected sheep between 8 and 19 wpi. In contrast, serum beta-HOB levels were elevated in F. hepatica infected sheep between 6 and 16 wpi and in F. gigantica infected sheep between 7 and 15 wpi. It would appear from these serum glucose and beta-HOB levels that fasciolosis does lead to energy deficiency (low glucose) and ketosis (increased beta-HOB). The decrease in serum glucose and increase in serum beta-HOB levels in infected sheep may help in understanding the interaction between fasciolosis and nutritional status of infected ruminants especially in young growing animals.     

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.     

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

The use of cysteine proteinases from Fasciola hepatica adult flukes for the serodiagnosis of caprine fasciolosis by means of an indirect ELISA test was studied. Two proteolytic fractions from adult fluke homogenates, with apparent molecular weights of 28 and 34 kDa (P28 and P34 respectively), were characterised as cysteine proteinases using azocasein assays and gelatin gel analysis. Both P28 and P34 fractions were electroluted and used as antigens in two different indirect ELISA tests. Serum IgG levels against P28 and P34 in goats given an experimental primary infection with 200 metacercariae or in goats given two experimental infections with 200 metacercariae were determined and compared with those observed in an uninfected control group. ELISA tests using both cysteine proteases showed a rapid and consistent detection of specific IgG in all experimentally infected goats. The IgG response to P28 was the first to be detected as early as 2-3 weeks post-infection and remained elevated throughout the experiment. The response to P34 was detected later (4-6 wpi) and disappeared in some animals at 18 wpi, while flukes were still present in the bile ducts. No significant differences were observed between the anti-P28 and anti-P34 IgG responses between animals receiving a primary or a challenge infection. The results of our study, although preliminary, are promising since the P28 ELISA described here may be a reliable method for the immunodiagnosis of F. hepatica infection in goats.     

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively.In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.     

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.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Fasciola gigantica excretory-secretory products for immunodiagnosis and prevention of sheep fasciolosis

Excretory-secretory products (ESP) products of ex vivo Fasciola gigantica adult worms were used for immunodiagnosis of sheep experimental infection with F. gigantica and natural infection with Fasciola spp. by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Specific IgG antibody binding to native or denatured ESP was detected as early as 2 weeks after experimental sheep infection with 100 or 200 metacercariae. No specific IgG antibody binding was displayed by sera obtained from 192 sheep considered to be Fasciola- and other parasite-free by microscopic examination of bile and feces. Additionally, sera from 200 apparently Fasciola-free sheep, yet infected with other parasites, were all negative. The data, thus, indicated that ESP-based ELISA reached nearly 100% sensitivity and specificity in immunodiagnosis of sheep fasciolosis. As expected, the ESP molecules were immunogenic in sheep eliciting interleukin-12p40 mRNA response and considerable amounts of antibodies, which were able to bind to the surface of newly excysted juvenile worms as judged by membrane indirect immunofluorescence, and mediate their attrition via antibody-dependent cell-mediated cytotoxicity. The ESP-induced cellular and humoral immune responses were associated with a modest reduction in worm count, yet with a highly significant (P<0.0001) decrease in size of recovered worms, thus suggesting that ESP immunization might be a safe and cost-effective strategy for reducing transmission of the infection.     

Analysis of the IgG antibody response against Paramphistomidae trematoda in naturally infected cattle. Application to serological surveys

The IgG antibody response to Calicophoron daubneyi (Digenea: Paramphistomidae) excretory/secretory antigens was evaluated in naturally infected cattle from Lugo (Galicia, NW Spain) by using an ELISA procedure. Two studies were conducted, first a survey in 524 cattle separated into three groups according to age, G-1 (0–2 years old), G-2 (3–5 years old) and G-3 (>6 years old). In the second study, three groups of cattle were employed: G-I, naturally infected; G-T, naturally infected and treated with oxyclozanide plus levamisole (Nilzan Plus^®); G-C, cattle maintained in a farm where C. daubneyi has never diagnosed. Variations on egg-output and haematic parameters (erythrocytes, haematocrite, leukocytes and lymphocytes) were also analyzed.

The ELISA procedure showed that 61.2% of the cattle in the first study had been exposed to the trematode, but only 10.1% passed eggs in the feces. Age-association with egg-output was shown but not with the IgG values. In the second experiment, the administration of the anthelmintic reduced significantly the IgG kinetic levels and the C. daubneyi-egg-output was suppressed during 12 weeks in the G-T group. The values of red cells, haematocrite, leukocytes and lymphocytes increased significantly in the treated cattle 5 weeks after chemotherapy; however, new reduction after week 5 was recorded, as results of the challenge of these cattle.

This is the first investigation in which evaluation of the IgG humoral response against C. daubneyi in cattle has been carried out. We proved that a notable IgG response in naturally infected cattle is induced, and can be detected by using an ELISA procedure. The IgG antibodies did not increase after challenge infection. Our results proved an important percentage of cattle were exposed to this trematode in the area of study and suitable measures for preventing this relationship must be considered.     

Detection of Fasciola hepatica antigen in cattle faeces by a monoclonal antibody-based sandwich immunoassay

A monoclonal antibody-based sandwich immunoassay (mAb sandwich ELISA) was developed for the detection of Fasciola hepatica antigen in the faeces of cattle. The assay was applied to samples from 100 cattle infected with F hepatica, 56 animals with parasitologically proven infections of other parasites and 100 uninfected animals. F hepatica antigen was detected in all the faecal samples from animals with fasciolosis, but none of the samples from the uninfected animals or from those with other parasitic infections had significant levels of F hepatica antigens. The results indicate that the mAb sandwich ELISA is a rapid, simple and useful method for the diagnosis of active F hepatica infection in cattle.     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological response of chickens to infection with Salmonella gallinarum-S. pullorum detected by enzyme-linked immunosorbent assay.

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.     

Characterization of the humoral immune response in alpacas (Lama pacos) experimentally infected with Fasciola hepatica against cysteine proteinases Fas1 and Fas2 and histopathological findings

A characterization of the humoral immune response of alpacas to Fasciola hepatica Fas1 and Fas2 antigens, two abundant cysteine proteinases in the excretory/secretory (E/S) products, was performed over the course of 6 months of experimental infection. Six adult alpacas aged 1-2 years old received a single dose of 200 F. hepatica metacercariae; two non-infected alpacas were kept as control group. All infected animals shed eggs 8 weeks post-infection (PI) and the number of flukes recovered at necropsy averaged 41+/-4. The livers of infected animals showed regions with chronic inflammation, granuloma containing parasite eggs, necrosis and cirrhosis. Peripheral eosinophilia in infected animals was greatly enhanced 6 weeks post-infection and later. A single peak of serum glutamic piruvic transaminase (SGPT) was observed 4 weeks PI and serum glutamic oxalacetic transaminase (SGOT) elevated 3 weeks PI and later. Circulating IgG Abs against Fas1 and Fas2 were measured by enzyme-linked immunosorbent assay (ELISA). Fas2-ELISA detected the infection 10 days PI reaching to highest titer on 7-8 weeks PI and kept elevated, until the end of infection. Fas1-ELISA detected the infection 2 weeks PI and followed the same pattern as Fas2-ELISA. Anti Fas2 IgG Abs were in higher titers and showed stronger avidity than anti Fas1 IgG Abs. In addition, rabbit IgG antibodies raised against cysteine proteinase Fas2 showed infiltration of this parasite antigen associated to the degradation of bile ducts and liver parenchyma of infected alpacas. In the present study we have established a F. hepatica experimental infection of alpacas, Fas2 appears to have a role in the pathogenesis of the liver damage in alpacas caused by the liver fluke. Infected alpacas elicited a strong humoral immune response against fluke cysteine proteinases Fas1 and Fas2, which might be considered as candidates for immunodiagnosis and vaccine development against fasciolosis in alpacas.     

Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle

AIM: To validate an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in sheep and cattle sera. METHODS: Gold-standard sera from sheep and cattle of known infection status, i.e. sera from non-infected animals and from animals known to be infected with F. hepatica were assayed with a commercially available ELISA and results analysed by ROC analysis. RESULTS: The ROC analysis suggested cut-offs that were considerably lower than those suggested by the manufacturer, yet the ELISA performed with high sensitivity and specificity, 98 to 100%, respectively for sheep and cattle sera. For bovine sera, particularly good discrimination between positive and negative sera was observed. Infection in experimentally infested animals could be demonstrated 7-8 weeks earlier than with classical parasitological techniques. CONCLUSIONS: The analysis of the ELISA's performance demonstrated high sensitivity and specificity. ROC analyses optimised the cut-off point suggested by the manufacturer of the commercial diagnostic assay. Diagnosis of infection with F. hepatica was achieved much earlier than is possible with current parasitological techniques. This could help with the control of fasciolosis, enabling treatment before clinical manifestation of the disease.     

THE EFFECT OF FASCIOLOSIS ON THE WOOL PRODUCTION OF MERINO SHEEP

The wool production of 20 Merino sheep, artificially infected with Fasciola hepatica, was compared with that of 20 uninfected controls. Sheep of two different ages, 6 months and 4 years, were fed in pens ad lib on two different diets giving high and low planes of nutrition. The midside tatooed patch technique was used to measure the wool production over periods of 6 weeks prior to the infection date, and 0–6, 6–12, 12–18 and 18–24 weeks after this date. Infection with F. hepatica caused a significant reduction of 20–39% in wool production from 6 weeks after infection, irrespective of the age of the sheep or the plane of nutrition. It was found that a reduction in wool production may occur without symptoms of fasciolosis being apparent.     

Use of avidity enzyme-linked immunosorbent assay and avidity Western blot to discriminate between acute and chronic Neospora caninum infection in cattle.

Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment.     

Infection and reinfection of chickens with Salmonella typhimurium: bacteriology and immune responses.

Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.     

A recombinant-based ELISA evaluating the efficacy of netobimin and albendazole in ruminants with naturally acquired fascioliasis

The therapeutic efficacy of albendazole and netobimin in ruminants with naturally occurring fascioliasis was investigated using a recombinant-based ELISA. The variation in the IgG response against a 2.9-kDa recombinant protein (FhrAPS), termed efficacy index (EI) 1, and the egg-output changes, termed EI 2, were used to evaluate drug efficacy.The values of EI 1 ranged between 0% and 50% in sheep, and between 0% and 30% in cattle after treatment with albendazole and netobimin. Similar EI 2 values were observed in sheep receiving albendazole or netobimin, but the highest values were found in cattle treated with netobimin. The significant reduction in the IgG response to FhrAPS found in this study shows promise in terms of developing alternative methods for evaluating the efficacy of chemotherapy against Fasciola hepatica in grazing ruminants.     

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.     

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.