Diagnosis of Fasciola sp. infections in cattle by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was evaluated for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle. The positive rate for the ELISA in calves inoculated with Fasciola metacercariae were 21.1% by 2 weeks postinoculation (PI), 94.6% by 4 weeks PI and 100% by 6-21 weeks PI. The positive rate for the immunodiffusion test (Ouchterlony test) reached 91.7% by 2 weeks PI, however, it dropped to 77.8% by 10 weeks PI. The positive rate for the fecal egg examination was 0% by 10 weeks PI, 77.8% by 12 weeks PI and 100% by 14-21 weeks PI. The practical application of ELISA was tested by using 165 cows raised under field condition. All the 24 cows that were positive both in the fecal egg examination and the Ouchterlony test were ELISA positive. Of the 6 cows that were egg positive and Ouchterlony negative, 5 showed ELISA positive reactions. Of the 27 cows that were egg negative and Ouchterlony positive, 24 were ELISA positive. Of the 108 cows that were egg negative and Ouchterlony negative, 90 were ELISA negative. However, the other 18 cows had ELISA positive reactions. Our results suggested that the ELISA using crude adult antigen was superior to the Ouchterlony test and fecal egg examination for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle.     

Herd evaluation of Fasciola hepatica infection levels in Louisiana cattle by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was evaluated as a method of determining relative immunoconversion rates in calves and how immunoconversion rates and strength of optical density values correlate with prevalence of Fasciola hepatica fecal egg shedding. Ten to 55 calves and cows were examined from each of 10 separate beef cattle herds in central and southern Louisiana. Infection prevalence rates for calves averaged 8% higher when ELISA optical density values were used than those when fecal egg count data were used. Of 55 calves in 8 herds that were ELISA positive, 39 were shedding F hepatica eggs; of 53 calves that were shedding eggs, 14 were ELISA negative. Significant correlation of calf fecal and ELISA prevalence was observed for 8 herds by linear regression analysis. A chi 2 analysis showed that calf ELISA and fecal egg shedding data were not independent. Results indicate that positive ELISA reactions for as few as 10 to 15 calves from the fall calf crop of a given herd are sufficiently accurate to be used to assess F hepatica herd infection rates, the likelihood of liver condemnations at feedlot destinations, and variation between individual farms in fascioliasis infection risk. The test was less valuable as a diagnostic test when used in adult animals previously exposed to F hepatica or on an individual animal basis.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica in milk

Antibodies against Fasciola hepatica were detected in serum and individual milk samples of dairy cattle using an ELISA. Percentage positivity (PP) values in milk samples were related to serum PP values and were not influenced by days into lactation. The correlation coefficient between serum and individual milk samples was highly significant (r=0.84, P<0.005). The correlation coefficient between herd seroprevalence and herd milk antibody prevalence was 0.96. The correlation coefficient between prevalence measured by faecal egg count and both seroprevalence and milk antibody prevalence within the herd was 0.87. The diagnostic sensitivity and specificity for milk were 92% (95% CI=89-96) and 88% (95% CI=85-91), respectively, when the serum test was considered as a gold standard. In conclusion, the level of antibody to F. hepatica in milk is significantly correlated with the antibody level in serum and this ELISA is suitable as a means of routine veterinary diagnosis of exposure to F. hepatica in cattle and an alternative to testing sera.     

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.     

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

AIM: To determine the performance characteristics of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in bovine milk. METHODS: Serum and milk from liver fluke infected and non-infected cattle was assayed in a commercially available enzyme-linked immunosorbent assay. Serum test results were used to determine the "gold standard" infection status of cattle and milk ELISA results assessed by ROC analysis. RESULTS: ROC analysis suggested changes to the ELISA protocol, arriving at milk dilutions assayed considerably higher than those suggested by the manufacturer. With those changes, the ELISA performed with high sensitivity and specificity, 95 and 98.2%, respectively, for individual bovine milks (relative to sera). For bovine tank milks, sensitivity was lower, with bulk milks only testing positive if 60% or more of cattle milking in the herd were infected. CONCLUSIONS: The analysis of the ELISA's performance when used on individual bovine milks demonstrated high sensitivity and specificity. ROC analyses optimised the assay conditions and cut-off point suggested by the manufacturer for this commercial diagnostic assay. This would help with the identification and control of fasciolosis, enabling simpler sample collection.     

Sero-diagnosis of Fasciola hepatica infections in cattle

An antigen was prepared from metabolic products which were produced by maintaining Fasciola hepatica adults in RPMI tissue culture medium for 24 h. The antigen compared favourably with a soluble extract of adult flukes when used in an enzyme-linked immunosorbent assay (ELISA). Experimentally infected calves were used to evaluate the sensitivity of the test and a survey from areas of both traditionally high and low incidence of the disease was carried out. Evidence is presented which suggests that this metabolic antigen could be used for the sero diagnosis of naturally occurring infections. In addition the use of a microcomputer to read, file and analyse the results enabled a very large number of samples to be processed daily.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.     

Diagnosis of Corynebacterium pseudotuberculosis infections in sheep, using an enzyme-linked immunosorbent assay

Lambs infected with Corynebacterium pseudotuberculosis (ovis) by injecting suspensions of live bacteria into the wool-free area of the axilla developed antibody against cell wall antigens and antitoxin, as measured by the enzyme-linked immunosorbent assay. The toxin was a better antigen for measuring infection than was the cell wall antigen. The enzyme-linked immunosorbent assay appeared to be as sensitive as the antihemolysin inhibition test for detecting antitoxin and was easier to perform.     

Efficacy of clorsulon against mature, naturally acquired Fasciola hepatica infections in cattle and sheep

Clorsulon (3.5 or 7 mg/kg of body weight) was given orally to mature cows (dairy or beef) and to mature mixed-breed sheep harboring patent infections of Fasciola hepatica. Eighteen animals of each species were assigned to a control group (drug vehicle) or to 1 of 2 treatment (3.5 or 7.0 mg/kg) groups of 6 animals each. On posttreatment days 8 (cows) or 14 (sheep), the animals were slaughtered for recovery of flukes. In cows, the efficacy (P values for treatment groups vs control) of clorsulon against infections of mature F hepatica was 99.21% (P less than or equal to 0.0065) at 3.5 mg/kg and was 100% (P less than or equal to 0.0039) at 7 mg/kg. In sheep, the efficacy was 93.33% (P less than or equal to 0.0104) at 3.5 mg/kg and was 100% (P less than or equal to 0.0039) at 7 mg/kg. These results indicate that clorsulon is a highly effective compound for the treatment of mature F hepatica in cows and sheep.     

Diagnosis of bovine cryptosporidiosis by an enzyme-linked immunosorbent assay

This paper describes an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of cryptosporidiosis. A monoclonal antibody with a high affinity against an oocyst antigen was used to set up the test. The efficiency of this assay was compared with that of the flotation test; 275 calf faecal samples were examined by the two methods. There was 96% agreement between the two tests. For the 11 conflicting samples, the two tests were repeated and a modified Ziehl-Neelsen staining was performed on faecal smears. All these 11 samples contained few oocysts, but only five and six of them were shown to be positive by the ELISA and flotation tests, respectively. The degree of sensitivity of the ELISA and flotation tests is comparable; samples heavily or moderately contaminated with oocytes are detected by both methods. This ELISA is reliable and never gives rise to false positive results. Nevertheless, as with the flotation test, the occasional case containing very few oocysts will not always be detected by this test. If necessary, very accurate diagnosis can be made by a staining technique or by a direct immunofluorescent assay. In veterinary medicine, the ELISA seems to be a method of choice; it appears to be a fast and reliable technique which could be used as a routine test for the detection of Cryptosporidium oocysts. Nevertheless the degree of sensitivity must always be borne in mind. There is no need for a microscopic examination, which is an additional advantage.     

Diagnosis of psoroptic sheep scab with an improved enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of specific antibodies against crude Psoroptes antigen. The diagnostic sensitivity was 93.7% in 191 sheep with clinical signs associated with mange. These animals originated from 29 flocks in which psoroptic mites were detected. All of 59 sheep infested with Psoroptes ovis were seropositive. Additionally, in 49% of 70 clinically unaffected sheep originating from P. ovis-infested flocks, specific antibodies could be detected, suggesting that asymptomatic infestations can be diagnosed by serology. The specificity of the ELISA was 96.5% as determined with 254 sheep originating from 44 flocks without clinical mange. Cross-reactivity in a low range was detected with selected sera of sheep with clinical chorioptic or forage mite infestations. Four sheep seroconverted 2 weeks after experimental P. ovis infestation, i.e. 2 weeks before clinical signs became obvious. After successful doramectin treatment of 14 sheep with naturally acquired P. ovis infestation, the ELISA values declined slowly but remained positive in seven cases beyond 17 weeks.     

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.     

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.     

Chemotherapy of infection with Fasciola hepatica in cattle.

Diamphenethide and a mixture of nitroxynil and hexachlorophane, which are effective against Fasciola hepatica in sheep, are effective in cattle if allowance is made for the slower rate of development and the more intense liver reaction which is associated with resistance. Adult parasites are mostly rejected around the time of maturity so that therapy in cattle must be directed against the early developmental stage using an appropriate drug.     

Serological diagnosis of cysticercosis by an enzyme-linked immunosorbent assay in experimentally infected cattle.

Taenia saginata infections were established in four groups of calves by administering doses of 10, 10(2), 10(3) and 10(4) infective eggs respectively by gavage. A fifth group remained as uninfected controls. Sera were collected from all calves over a period of 210 days. The sera were examined by an enzyme-linked immunosorbent assay (ELISA) with a fraction of larval Taenia hydatigena cyst fluid as antigen for the presence of anti-T. saginata IgG antibodies. At slaughter, the tongue, masseter, diaphragm and cardiac muscles and liver were examined for cysticerci. The higher dose rates of T. saginata eggs were reflected in higher numbers of cysticerci found in the calves at necropsy. There was also a correlation between higher levels of antibodies produced as measured by the ELISA and the numbers of eggs given. Sero-conversion was first detected about 25 days postinfection in heavy infections and later in the lighter infections. Maximal levels of antibody occurred between 40 and 60 days postinfection, followed by a gradual decrease in levels of antibody. A secondary increase in antibody occurred between 160 and 200 days postinfection which might have been due to release of antigen after death of the cysticerci. The low level of circulating antibodies in light infections may result in false positive or false negative diagnoses depending upon the selection of the cut-off point.     

Comparative evaluation of the enzyme-linked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle

The enzyme-linked immunosorbent assay (ELISA) was evaluated for the diagnosis of naturally acquired Fasciola gigantica infection in cattle in comparison with conventional parasitological techniques. Using unfractionated whole worm extract of F. gigantica as the antigen, it was observed that there was a good correlation between ELISA positivity and positive diagnosis of fascioliasis by post mortem liver examination, bile egg sedimentation and faecal egg sedimentation. There was, however, a disparity between ELISA results and faecal egg flotation results.     

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.