Indirect Hemagglutination Technique Research Articles

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Seroprevalence of fascioliasis in the eastern region of Turkey: an eight-year investigation.

Fascioliasis is a zoonotic disease and one of the most neglected infectious diseases in humans. Its prevalence has been increasing significantly during the last decades. This study aimed to investigate the prevalence of fascioliasis using direct microscopy and indirect hemagglutination (IHA) technique in a region in Eastern Anatolia of Turkey. This study was conducted on the serum samples obtained from 817 patients (372 male and 445 female) between 2011 and 2018, who were suspected to have fascioliasis. IHA was used to investigate anti-Fasciola hepatica antibodies in the serum samples. Stool specimens were obtained from the seropositive patients and were examined with the native-Lugol method to identify the parasites. It was determined that 5.5% (45/817) of all the patients were F. hepatica seropositive and 6.4% (52/817) were borderline positive. Positivity was 5.7% (21/372) among males and 5.4% (24/445) among females, and the difference in the infection rates between these groups was not significant (p=0.913). The highest number of patients who applied to the clinic was in the "45 and over" age group (317 patients); 270 patients were in the 25-44 age group. A maximum positivity of 10.3% was observed in the 7-14 age group. Previously, fascioliasis was considered a rare infection in humans; however, it has emerged as an important public health problem in the world. Considering fascioliasis in patients with clinical symptoms, not only with direct observation but also using serological methods, would be effective in early diagnosis and treatment of the disease.

Trypanosoma cruzi seroprevalence and associated factors in women in Casanare-Colombia.

Chagas disease is caused by the parasite Trypanosoma cruzi and transmitted mainly by triatomines and from mothers to children. In Colombia, this disease is a public health problem and due to its high endemicity and vertical transmission, women are susceptible populations that must be evaluated. Our objective was to determinate the Trypanosoma cruzi seroprevalence and factors associated with women in Pore (Municipality), Casanare, Colombia. Cross-sectional study. A sample of 230 healthy volunteer women, 15years or older, without previous diagnosis of Chagas disease was taken; the serological analysis was done using the Chagas ELISA IgG and IgM and indirect Hemagglutination (HAI) technique. In addition, a survey was applied to each participant in order to explore the presence of factors that could be associated with a positive test result. The seropostitivity found in Pore Casanare's women was 16.9% (39/230, 95% CI 12.1-21.7), additionally it was found that rural origin, the coexistence with animals, especially chickens, age, low level schooling and housing material are factors associated with T. cruzi infection in this population. The results of this study indicate the importance of conducting extensive seroepidemiological studies in populations of endemic areas, due to the difficulty in detecting cases in the acute phase; therefore, screening allows the establishment of a follow-up and treatment time line that contributes to the interruption of the transmission vertical.

SEROPREVALENCE OF TOXOPLASMA GONDII IN DOGS AND CATS DOMICILED IN THE WEST OF SANTA CATARINA, BRAZIL

Toxoplasma gondii is a protozoan with worldwide distribution, responsible for causing toxoplasmosis, a disease that affects mammals, reptiles, amphibians and birds. The objective of this study was to evaluate T. gondii seroprevalence in dogs and cats domiciled in cities from West and Serrana mesoregions of Santa Catarina. A total of 338 blood samples, 211 from dogs and 127 from cats were collected and submitted to the indirect haemagglutination technique (IHT) for the detection of anti-T. gondii antibodies. Epidemiological questionnaire asking for information related to gender, breed, temperament, behavior, immunity, body index, diet, ectoparasites, access to the streets, hunting habit was applied to the animals owners. The results were 71.89% (243/338) of seropositive animals, being 69.19% (146/211) dogs and 76.38% (97/127) cats. The variable that presented statistical significance was commercial ration for dogs as protection factor. The present study demonstrates a high prevalence of anti-T. gondii antibodies in dogs and cats domiciled in West and Serrana mesoregions of Santa Catarina state, Brazil. Through the obtained results we can reinforce the importance of feeding dogs with commercial food, rather than home-made food, avoiding the consumption of raw or undercooked meat by the animals.

Detection of Toxoplasma gondii infection in feral wild boars (Sus scrofa) through indirect hemagglutination and PCR

ABSTRACT: Wild boars (Sus scrofa) have become an important invasive species in all Brazilian regions. Increase in their population causes damage to rural properties, as they invade and destroy crops. To protect their crops and farm animals, producers hunt wild boars and often consume the product without any sanitary control, becoming exposed to various types of pathogens, including Toxoplasma gondii. Sanitary evaluations of these animals are scarce, especially in relation to the protozoan T. gondii. This study aimed to evaluate the occurrence of this pathogen in wild boars in Brazil. We analyzed 122 blood samples from wild boars (blood clots and serum), collected between 2014 and 2016 in five Brazilian states, using polymerase chain reaction (PCR) and indirect hemagglutination (IH) techniques. In total, 33 (27%) samples were positive by at least one test, 16 (13.1%) were positive by PCR, 19 (15.6%) were positive by IH, and only 2 (1.6%) were positive by both tests. The lack of sanitary management of feral animals increases the incidence of infections, and the consumption of raw or inadequately cooked meat may become a potential source of infection for humans in Brazil.

Prevalence of RhD variants among blood donors at Gulu Regional Blood Bank, Gulu, Northern Uganda.

Aim/objectiveThe aim of this study was to determine the prevalence of RhD variant phenotypes among voluntary non-remunerated blood donors (VNRBDs) at Gulu Regional Blood Bank (GRBB), Northern Uganda.Materials and methodsWe conducted a cross-sectional study, in which the first 4.0 mL of ethylenediaminetetraacetic acid (EDTA) blood samples were collected from VNRBDs and typed for their ABO and RhD blood group status using IgM and IgG monoclonal typing antisera, respectively. Blood samples that tested as RhD negative were further investigated for RhD variant phenotypes using indirect antihuman globulin hemagglutination technique.ResultsWe assayed 138 RhD-negative blood samples obtained from VNRBDs. Of these, 66.7% (n=92) were males. Their median age was 24.4 years (range, 14–33 years). Majority of the participants were of ABO blood group O (62.8%, n=86), followed by A (19.7%, n=27), then B (13.9%, n=19) and least AB (3.6%, n=6). The prevalence of RhD variant phenotypes was 0.7% (n=1; 95% confidence interval, 0.5–0.9). There was no statistical association of RhD variant phenotypes with donor gender, tribe and their ABO blood groups.ConclusionThis study has revealed a high prevalence of RhD variant among blood donors at GRBB in Northern Uganda. It further highlights a potential risk of alloimmunization, as the present blood typing practices do not identify RhD variant phenotypes.

A WHO reference reagent to standardize haemagglutination testing for anti-A and anti-B in serum and plasma: international collaborative study to evaluate a candidate preparation.

The aim of the study was to evaluate a lyophilized serum preparation, 14/300, for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize and control haemagglutination titrations for anti-A and anti-B in serum and plasma, in an international collaborative study. Serum preparation 14/300 and two plasma-based reserve preparations, 14/304 (high titre anti-A) and 14/208 (high titre anti-B), were titrated by 24 laboratories in 13 countries using direct (DRT) and indirect (IAT) haemagglutination techniques. There was eightfold to 64-fold variation in reported titres per preparation and method across laboratories, that is, titres extended over 4-7 dilutions, although intralaboratory variability was generally good, with over 90% of replicate titres within a twofold range. There was a reduction in interlaboratory variability when titres of the reserve preparations were adjusted relative to those of the candidate Reference Reagent. The establishment of 14/300 as a WHO Reference Reagent for high titre anti-A and anti-B in serum, with nominal anti-A and anti-B titres of 128 for DRT, and nominal anti-A and anti-B titres of 256 for IAT, will facilitate global standardization of haemagglutination titrations for anti-A and anti-B in patient samples and blood components.

Indirect Heamagglutination as an Immunodiagnostic Technique for Cystic Echinococcosis in Iraqi Patients

Objective: The aim is to determine the effectiveness of indirect Haemagglutination (IHA) as diagnostic techniques for Cystic Echinococcosis in Iraqi and the possible correlation of age and gender in disease occurrence. Material and Methods: IHA technique using sensitized sheep red blood cells coated with an Echinococcus granulosus antigen. A chi square, Pearson correlation used for analysis of categorical variables. Results: Patients presented with clinical manifestation of hydatidosis (202) selectively enrolled in this study. The mean age was 31.64±8.98 years. The minimum age was 18 years and the maximum age was 70 years. Males represent 50%, with the mean age of 33.50±7. 8 years. The rest of patients were females with a mean age of 29.78±9. 6 years. Positive cases represent 19.3%, and the 80.7% were negative. The number of positive cases was higher among females (22.8%) compared with males (15.8%) with no significant difference (p= 0.212) or correlation between gender and IHA results (p = 0.214) .The majority of patients had a the rage of 24-30 years, 31-36 years and 18-23 years. Among males, a higher number of positive cases at 31-36 years followed by 24-30 years and 49-54 years. Among females, a higher number of positive cases with 24-30 years of age was followed by those with 37-42 years of age and 18-23 years of age. There was a significant difference (p= 0.001) between age groups according to gender without a significant correlation between the age group and IHA results according to gender (p= 0.268). Conclusion: The IHA is considered as rapid, reliable and adequate technique that makes it possible to process a large number of samples simultaneously. It does not require highly trained technical personnel. Females appear to be more susceptible to CE than males especially in younger age groups.

Serotyping and identification of predisposing factors for the occurrence and distribution of ovine pasteurellosis in Bonga sheep breed

Cross-sectional study for investigating the prevailing field serotypes and predisposing factors for the occurrence and distribution of ovine pasteurellosis in Bonga sheep breed of different agro-ecology of Adiyo (highland), Gawata (lowland) and Debub Bench (Midland) of Kaffa and Benchi Maji in southern Ethiopia was conducted during July 2012 to June 2013. For this purpose, a questioner survey to identify predisposing factors and laboratory analysis on samples to investigate the prevailing field serotypes were performed. In these regard, a total of 210 farmers were interviewed and 70 blood samples from each agro-ecology (total of 210) was randomly collected following standard procedures. Laboratory analysis was performed by indirect haemagglutination (IHA) technique at National Veterinary Institute (NVI), Debre Zeit, Ethiopia. The finding showed that, 34.8% of the farmer’s household main sources of income were dependent on sheep production in which ovine pasteurellosis had 81% of occurrence. Management of sheep like housing and sanitation of the barn were predisposing factors. Irrespective of agro-ecology, M. haemolytica A2 (52.4%) followed by A7 (42.4%) and A1 (31%) were detected where as P. multocida serotype was not detected. Among identified serotypes, highest prevalence of M. haemolytica A1 (37.1%) were detected in lowland where as A2 (62.9%) in Midland and lowland with equal prevalence and A7 (85.7%) in midland. Therefore, ovine pasteurellosis was the major disease of sheep identified with multiple losses in the production and M. haemolytica is the most common cause identified. Measures such as, improving management practices by providing optimal sanitation in housing, providing good quality supplement feeding during rainy season and minimizing stress factors should be taken into account to reduce the prevalence. In addition, the currently use vaccine (P. multocida biotype A) not incorporate the identified serotypes so that may not protect the disease. As a result, use of multivalent vaccine by incorporating those identified serotypes is necessary to effectively prevent the disease.Asian J. Med. Biol. Res. June 2015, 1(2): 175-181

Modification of a Commercial Toxoplasma Gondii Immunoglobulin G Enzyme-Linked Immunosorbent Assay for use in Multiple Animal Species

A challenge faced by veterinary diagnosticians in serologic analysis for exposure to pathogens is the need for a protein conjugate capable of antibody attachment in many animal species. The advent of protein conjugates that are less specific in nature allows diagnosis across many species with little or no modification of technique. Toxoplasma gondii is an organism of veterinary interest that has been demonstrated to infect a plethora of warm-blooded animals. However, the serologic tests available for simultaneous diagnosis in this broad range are limited in number. The current study examined the use of an immunoglobulin G enzyme-linked immunosorbent assay (ELISA) modified by the use of non-species-specific protein conjugates in domestic animal species commonly submitted to diagnostic laboratories for evaluation of Toxoplasma exposure status. Comparison with results from an established indirect hemagglutination technique revealed very good agreement between the 2 test methods. This modification of the ELISA provides a useful method for veterinary diagnosticians to perform rapid and accurate evaluation of multiple animal species for Toxoplasma exposure using a single test.

Seroepidemiology of toxoplasmosis in women of childbearing age from a marginal community of Maracaibo, Venezuela

The aim of this study was to determine the prevalence of anti-T. gondii total and IgM antibodies in women of childbearing age. One hundred serum samples of women were studied with age range from 11 to 45 years old. Samples were chosen by random. The determination of total antibodies was carried out through the indirect hemagglutination technique and IgM antibodies by ELISA's technique. The statistical analysis was carried out through the Chi square and the Spearman correlation tests. The theoretical estimated incidence of congenital toxoplasmosis was calculated, according to the annual increment of antibody prevalence among the age groups. The overall prevalence of toxoplasmosis was 33%, while only six individuals (18.2%) were positive to IgM. The highest prevalence was observed in the 11-35 year-old age group. The theoretical estimated incidence was 1.5 for 100 pregnancies in women of 21-25 year-old group; it decreased until 0.1% in the 41-45 year-old age group. The findings show a high prevalence of toxoplasmosis in this community with a high infection risk in women of the studied age group and the high cat population observed, suggesting that the transmission way by contaminated soils may play a main role in the spreading of toxoplasmosis in this community.

Expression of complement C3b/C4b receptors (CR1) on erythrocytes from patients with myasthenia gravis

The complement C3b/C4b receptor (CR1) expression on erythrocytes (E) (ECR1) from patients with myasthenia gravis (MG) and from blood donors was measured using haemadsorption and indirect hemagglutination techniques. The ECR1 expression in patients with MG appeared to be normal. This was also true for the subgroup of MG patients with additional autoimmune diseases. No correlation between ECR1 expression and severity of myasthenic symptoms was demonstrated.

A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response was tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinssetics were confirmed in susceptible mice, with peak IgG response seen ∼8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.

Echinococcus granulosus in jordan: assessment of various antigenic preparations for use in the serodiagnosis of surgically confirmed cases using enzyme immuno assays and the indirect haemagglutination test

The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB), and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation ( r = 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24 KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen B fractions of CSHF or AAB are detected.

An epidemiological study of Fasciola hepatica in The Netherlands.

Transmission of F. hepatica under natural conditions was analysed in a three year programme. The variables used were the indirect haemagglutination (IHA) technique, worm establishment in tracer lambs and the population dynamics, infection rate and shedding pattern of Lymnaea truncatula. It is concluded that fluke eggs, infected snails and metacercariae on herbage can survive the winter in the Netherlands. Metacercarial availability was positively correlated to the amount of rainfall in the grazing period. The role developed eggs that survive the winter is important, because this results in earlier infections in the herd. The use of the serological diagnosis method IHA is important to detect F. hepatica infection in an early stage. Use of cellophane paper on floats is a useful method for determining the shedding pattern of cercariae from L. truncatula. It is concluded that collection of metacercariae on cellophane floats, inventarization of L. truncatula and its infection level are useful tools for the prediction of liverfluke infections.

Serological response to tetanus toxoid: a field study in Pondicherry, India

To study the serological response to various doses of tetanus toxoid given to pregnant women, 320 samples of blood obtained from 173 pregnant women were analysed using the indirect haemagglutination technique. Two doses of toxoid were necessary to achieve protective titres in women who were previously unimmunized. The antibody levels appeared to persist for up to 4 years. During a subsequent pregnancy, a single booster dose of toxoid was sufficient to raise the titres adequately for protection. These findings are in accordance with the immunization programme followed for prophylaxis against tetanus among pregnant women.

Acute Chagas' disease ( Trypanosomiasis americana) in acquired immunodeficiency syndrome: Report of two cases

Two heterosexual men, aged 31 and 40 years, with the acquired immunodeficiency syndrome and presenting with the acute form of Chagas' disease are reported. The first patient, a carrier of hemophilia A, was treated for 20 years with Chilean and Brazilian cryoprecipitates. This patient acquired both diseases through this medium. The second patient, an inhabitant of northern Chile (fourth region), was allegedly bitten by Triatoma infestans and was an intravenous drug addict. The hemophilic patient presented with a neurologic syndrome; a brain biopsy showed a necrotizing encephalitis with an obliterative angiitis and abundant macrophages. The second patient developed intractable congestive heart failure; necropsy showed a dilated myocarditis with rupture of myofibers and an inflammatory infiltrate rich in plasma cells, lymphocytes, and macrophages. Using light and electron microscopy, abundant amastigotes of Trypanosoma cruzi were seen in brain tissue, especially in the cytoplasm of macrophages, as well as in some myocardial fibers. In both cases, determination of anti-T cruzi antibodies (indirect hemagglutination technique) and xenodiagnosis were positive.

Comparison of Latex Agglutination, Indirect Hemagglutination, and ELISA Techniques for the Detection of Toxoplasma gondii‐specific Antibodies in the Serum of Cats

The primary purpose of this study was to determine whether commercially available latex agglutination and indirect hemagglutination kits for the detection of Toxoplasma gondii-specific antibodies were capable of detecting T. gondii-specific immunoglobulin M (IgM) in the serum of cats. Serum samples from 35 cats containing either T. gondii-specific IgM, T. gondii-specific immunoglobulin G (IgG), or both were collected. Each serum sample was assayed using a latex agglutination kit, an indirect hemagglutination kit, an enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii-specific IgG, and an ELISA for the detection of T. gondii-specific IgM. When serum samples containing only T. gondii-specific IgM as determined by ELISA were assayed, the latex agglutination kit and the indirect hemagglutination kit detected antibodies in 33.3% and 13.3%, respectively. When T. gondii-specific IgG was present in a serum sample, the results from the latex agglutination kit, the indirect hemagglutination kit, and the IgG-ELISA were similar; however, there was a wide variation in titer magnitude results between the three assays. It was concluded that the latex agglutination kit and the indirect hemagglutination kit did not adequately detect T. gondii-specific IgM in feline serum.

Circulating diphtheria antitoxin levels in children aged 11–14 years in relation to the vaccinal history

Diphtheria antitoxin level in serum samples obtained from 204 healthy children aged 11–14 years was determined by means of an indirect haemagglutination technique and related to the vaccinal history of the subjects. Irrespective of the time since the last toxoid inoculation, the mean antitoxin titre per ml of serum in the individuals who had received incomplete/irregular anti-diphtheria vaccination in childhood was significantly higher when the number of toxoid doses was higher (from two to more than four doses); the same was not observed for individuals given primary vaccination (three toxoid doses) according to the schedule for childhood vaccination in Italy (regular vaccines) and one or more booster doses. Between 8 and 13 years after the last of three toxoid doses, a significantly negative association between mean antitoxin titre and time was observed only in irregularly vaccinated children ( r= −0.82; p < 0.05); nevertheless, up to thirteen years after the last vaccine dose, more than 95% of the children exhibited protective levels of antitoxin (> 0.125 turkey red blood cells passive haemagglutination units per ml). No significant decrease in mean antitoxin titre was observed between 4 and 10 years after the last of four either regularly or irregularly administered toxoid doses.

A new method for serodiagnosis of sheep fascioliasis using helminth excretory-secretory products

The diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) for antibodies to Fasciola hepatica was evaluated using adult fluke excretory-secretory products as antigen. The sensitivity and specificity of the test were compared with those of the indirect hemagglutination (IHA) technique. The results of the DIG-ELISA showed that the reaction zone diameters obtained with 42 sera from sheep with natural or experimental exposure to F. hepatica were significantly greater than those obtained with 85 sera from ovines infected with other parasites. Both sensitivity and specificity were 100% in the DIG-ELISA, whereas the sensitivity was 68.2% and the specificity was 100% in the IHA. The data suggest that the DIG-ELISA is a valuable serodiagnostic test for sheep fascioliasis.