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Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Comparative evaluation of the enzyme‐linked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle a
B. O. Fagbemi & I. O. Obarisiagbon
a
a
Department of Veterinairy Microbiology and Parasitology , University of Ibadan , Ibadan, Nigeria Published online: 01 Nov 2011.
To cite this article: B. O. Fagbemi & I. O. Obarisiagbon (1990) Comparative evaluation of the enzyme‐linked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle, Veterinary Quarterly, 12:1, 35-38, DOI: 10.1080/01652176.1990.9694239 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694239
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Comparative evaluation of the enzymelinked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle
Downloaded by [University of South Florida] at 12:22 06 November 2014
B. 0. Fagbemi and I. 0. Obarisiagbonl SUMMARY 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.
INTRODUCTION
In cattle that harbour patent infections of Fasciola, the recovery of eggs from their faeces remains the surest method of diagnosis of liver fluke infection in spite of the vast array of serodiagnostic techniques that have evolved and been described over the past two decades (11). Apart from their inability to detect prepatent infections, the cumbersomeness and the apparent lack of sensitivity of conventional coprological methods coupled with the need for simple, rapid and sensitive diagnostic techniques that are applicable to epidemiological investigations have justified the search for reliable serotests. One serodiagnostic test that has been adapted for the diagnosis of fascioliasis more recently is the enzyme-linked immunosorbent assay (ELISA) (1, 2, 3, 4, 5, 6, 9). In most of these works, ELISA was used for the diagnosis of Fasciola hepatica infection in experimentally infected animals. Furthermore, despite the elaborateness of some of these experimental designs which used different antigen types and protein fractions of E hepatica, there is still a need to truly assess the reliability of ELISA as a diagnostic tool in natural fascioliasis in relation to conventional parasitological techniques especially for E gigantica infection in African cattle. In this study, the sensitivity of ELISA in the diagnosis of naturally occurring E
gigantica infection in cattle was compared to conventional parasitological techniques.
MATERIALS AND METHODS
All the cattle studied were slaughtered at the Ibadan Municipal Abattoir, Nigeria. The age of the animals at the time of slaughter was 2 to 3 years. Sample collection: As described below, samples were collected from four groups of 688 slaughtered cattle: Group I: Blood samples were collected after slaughter from the hearts of 44 cattle whose livers were observed to be infected with E gigantica and from another set of 44 cattle whose livers were fluke-free. I
B. 0. Fagbemi and I. 0. Obarisiagbon, Department of Veterinairy Microbiology and Parasitology, University of Ibadan, lbadan, Nigeria.
THE VETERINARY QUARTERLY, VOL
12, No. I,
JANUARY 1990
35
Downloaded by [University of South Florida] at 12:22 06 November 2014
Group 2: Two hundred blood samples were collected randomly from cattle during slaughtering; their corresponding faecal samples were collected directly from the rectum. Group 3: Two hundred blood samples and faecal samples were also collected randomly from another group of cattle at slaughter. Group 4: In addition to blood and faecal samples, bile samples were randomly collected from the gall bladders of 200 cattle at slaughter irrespective of whether the livers were Fasciola-infected or Fasciola-free. Investigation procedure: ELISA was conducted on all the sera obtained from the four groups of animals. Foetal calf serum was used as Fasciola-free control serum. For the ELISA, E gigantica was prepared by homogenising 20 freshly collected adult flukes in 10 ml of phosphate buffered saline (PBS pH 7.2). The homogenate was left overnight at 4°C before it was centrifuged at 12,000 rpm for one hour at 4°C. The supernatant was stored at 4°C until used. A chequerboard titration (8) was initially performed to determine the optimum antigen concentration for adsorption to the plates and the optimum dilution of both the serum
and the conjugate. The procedure followed for the conduction of the ELISA was a modification of that of Burden and Hammet (2). The antigen was diluted in a carbonate buffer (pH 9.6) to give a solution containing 10
mg/m1; 100 pl of the solution was allowed to adsorb into the wells of polystrene microtitration plates for two hours at 37°C with one minute of shaking at 40-minute
intervals. The wells were washed 12 times with PBS containing 0.05% Tween 20, after which
the serum samples, diluted at a ratio of 1:100 with PBS, were added. The plates were incubated at 37°C for one hour. After washing again as described previously, peroxidaselabelled anti-bovine immunoglobulin (diluted at a ratio of 1:2000 with PBS) was added and the plates were incubated at 37°C for one hour with shaking after 30 minutes. The plates were washed once more and the enzyme substrate orthophenylene diamine, diluted I mg/m1 in 0.05% citrate buffer (pH 5.5), was added. After optimum colour development was allowed for 30 minutes, the reaction was stopped by the addition of 1M sulphuric acid. The optical density was determined spectrophotometrically at 405nm. The tests for all samples were run simultaneously. At the conclusion of the assays, the optical density readings for each group, including that for the negative control, were compared
to detect significant differences between group means. P values less than 0.05 were
considered significant. Particular cut-off points were not used. Parasitological studies: The faecal samples obtained from Group 2 animals were examined for E gigantica eggs by the sedimentation method (10), while those of Group 3 animals
were examined for fluke eggs by the zinc flotation method (7). In Group 4, both the sedimentation and flotation methods were used for the detection of the eggs from each
faecal sample. The bile collected from each animal in this group was examined for Fasciola eggs by sedimentation. For this, 25 ml of bile was centrifuged at 3,000 rpm for 30 minutes and the sediment was examined for Fasciola eggs. Study plan: The study conducted with Group I was designed to compare ELISA results for animals that were observed to be Fasciola-infected and Fasciola-free during post mortem examination. The purpose of the Group 2 studies was to compare the ELISA results with those obtained for the diagnosis of fascioliasis by the sedimentation method, while the purpose of the Group 3 studies was to allow a similar comparison with the flotation
technique. With Group 4, a simultaneous comparison of the four methods (ELISA,
sedimentation, flotation and post mortem diagnosis) was made. In Groups 2 and 3, samples were obtained from different sets of animals to broaden the
sample base and to obtain an independent comparison prior to the simultaneous comparison that was made with Group 4. RESULTS
The ELISA optical density (0D405) readings in the groups of cattle are presented in Tables 1 and 2. In Table 1, the OD readings obtained for cattle with Fasciolainfected livers (0.512 ± 0.181) was significantly higher than those for animals whose livers were Fasciola-free (0.135 ± 0.045). Similarly in Table 1, the ELISA result for sera of animals whose faeces contained 36
THE VF1TRINARY QIJARTFRIN,
Vol. 12, No 11 JArly
Table I. Antibody response to E gigantica infection in three groups of cattle as measured by ELISA (OD 405 nm ).
GROUPS Method of parasitological dia gnosis Liver examination
2 Egg sedimentation
Egg flotation
ELISA OD in sera of cattle positive for Fasciola according to method:
0.512 ± 0,181
0.424 ± 0.031
0.492 ± 0.170
ELISA OD in sera of cattle negative for Fasciola according to method:
0.135 ± 0.045
0.206 ± 0.167
0.416 ± 0.101
Downloaded by [University of South Florida] at 12:22 06 November 2014
1
3
ELISA OD in foetal calf serum = 0.104 ± 0.002
Fasciola eggs as detected by the sedimentation technique (0.424 ± 0.031) was significantly higher than for the cattle without eggs in faeces (0.206 ± 0.167). However, as shown in the same table, the OD values for cattle that were known to be Fasciola-infected as revealed by egg flotation (0.492 ± 0.170) was not significantly different from those of Fasciola-free cattle (0.416 ± 0.101). A simultaneous comparison of ELISA OD values for cattle that were ascertained
to be Fasciola-infected or Fasciola-free by bile egg sedimentation, faecal egg sedimentation and faecal egg flotation is shown in Table 2. It can be seen that while OD values for cattle that were proven to be Fasciola-infected by bile egg sedimentation and faecal egg sedimentation were significantly higher than those for animals that were Fasciola-free, the OD values for the same animals that were categorised by faecal egg flotation were not significantly different from each other. Table 2. Simultaneous comparison of ELISA OD values in a group of cattle in relation to parasitological diagnosis of E gigantica.
Method of parasitogical diagnosis 1
2
3
Bile egg
sedimentation
Faecal egg sedimentation
Faecal egg flotation
ELISA OD in sera of cattle positive for Fasciola according to method:
0.601 ± 0.034
0.462 ± 0.110
0.510 ± 0.061
EL1SA OD in sera of cattle negative for Fasciola according to method:
0.217 ± 0.052
0.214 ± 0.047
0.453 ± 0.059
ELISA OD in foetal calf serum = 0.101 ± 0.005 DISCUSSION
The results from these studies reveal that ELISA results for Fasciola-infected cattle were significantly higher than for the Fasciola-free animals when the methods used for determining the Fasciola-infection was liver examination, bile sedimentation
or faecal egg sedimentation. The difference in OD readings for the Fasciolainfected and the Fasciola-free animals was significant enough to justify positive diagnosis by the serological test. However, reliable results were not obtained when ELISA was used in relation to diagnosis of fascioliasis by faecal egg flotation. Illf tITIOolfor (1111010hr rill (1 it I
I
1
p
)1,
5.
)j.)
l.
,-
timR
11
Downloaded by [University of South Florida] at 12:22 06 November 2014
The equivocal results obtained in this latter case reveal the insensitivity of the faecal flotation method in the diagnosis of bovine fascioliasis. For instance simultaneous comparison of bile sedimentation, faecal egg flotation methods showed that many animals that were classified as Fasciola-free by faecal egg flotation were actually revealed to be Fasciola-infected by the two other methods. It was also observed during this study that zinc sulphate solution, because of its high specific gravity, caused considerable physical distortion of fluke eggs during flotation. Since the main aim of this investigation was to compare ELISA (OD) readings for the different groups 'of animals and not to establish strict positive or negative ELISA values, cut-off values were not set. The present findings confirm the usefulness of ELISA for the diagnosis of natural E gigantica infection as it had been shown for experimental E hepatica infection in cattle (2, 4) and sheep (11). These investigations have not taken into consideration the complications that may be caused by concurrent helminth infections with which E gigantica may share common antigens. In this connection. although Zimmerman et al. (11) demonstrated that concurrent nematode infections did not interfere with ELISA results obtained for experimental ovine fascioliasis, it is possible that antigens shared with more closely related trematodes such as schistosomes may affect the reliability of the test. Furthermore, the present studies have shown that reliable results may be obtained with the use of whole worm extracts as the antigen source. However, in view of the demonstration (9) that fractionated antigens revealed different stages of Fasciola infection in experimental fascioliasis in cattle, the use of fractionated antigens for diagnosing natural fascioliasis in cattle may produce more informative epidemiological data. ACKNOWLEDGEMENTS
This study was supported by the Danish International Development Agency (DANIDA), Denmark. REFERENCES
I. Ambroise-Thomas P, Desgeorges PT, Bouttaz M. Le diagnostic immunoenymologique (ELISA) de la humaine el bovine. Detection d'anticorps ef/ou d'antigenes circulants. Ann Soc Med Trop 1980; 60: 47-60.
2.
Burden DJ, Hammet NC. Microplate enzyme-linked immunosorbent assay for antibody to
Fasciola hepatica in cattle. Vet Rec 1978; 103: 158.
3. Burden DJ, Hammet NC. The ELISA test for detection of Fasciola hepatica infection in cattle. Parasitology 1979; 79: 3.
4.
Farrell CJ, Shen DT, Wescott RB, Lang BZ. An enzyme-linked immunosorbent assay for diagnosis of Fasciola hepatica infection in cattle. Am J Vet Res 1981; 42: 237-40.
Hillyer GV, Santiago de Weil N. Use of immunologic techniques to detect chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme-linked immunosorbent assay in infected rats and rabbits. J Parasitol 197; 65: 680-4. 6. Levine DM, Hillyer GV, Flores SI. Comparison of counterelectrophoresis, the enzyme-linked immunosorbent assay and kato faecal examination for the diagnosis of fascioliasis in infected mice and rabbits. Am J Trop Med Hyg 1980; 29: 602-8. 7. Manual of veterinary parasitological laboratory techniques. London (Technical bulletin n6. 18). Ministry of Agriculture, Fisheries and Food, 1971. 8. McLaren ML, Lillywhite JE, An ACS. Indirect enzyme-linked immunosorbent assay (ELISA): practical aspects of standardieation and quality control. Medical Laboratory Sciences 1981; 38: 5.
245-51.
9. Oldham G. Antibodies to Fasciola hepatica antigens during experimental infections in cattle 10.
measured by ELISA. Vet Parasitol 1983; 13: 151-8. Parfitt JW, Banks AW. A method for counting Fasciola eggs in cattle faeces in the field. Vet Rec 1977; 87: 180-2.
11. Zimmerman GL, HJ en LW, Cerro JE, Farasworth BS, Wescott RB. Diagnosis of Fasciola hepatica infections in sheep by an enzyme-linked immunosorbent assay. Am J Vet Res 1982; 43: 20972100.
38
THE VETERINARY QUARTERLY. VOL. 12, No. 1, JANUARY 1990
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Comparative evaluation of the enzyme‐linked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle a
B. O. Fagbemi & I. O. Obarisiagbon
a
a
Department of Veterinairy Microbiology and Parasitology , University of Ibadan , Ibadan, Nigeria Published online: 01 Nov 2011.
To cite this article: B. O. Fagbemi & I. O. Obarisiagbon (1990) Comparative evaluation of the enzyme‐linked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle, Veterinary Quarterly, 12:1, 35-38, DOI: 10.1080/01652176.1990.9694239 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694239
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Comparative evaluation of the enzymelinked immunosorbent assay (ELISA) in the diagnosis of natural Fasciola gigantica infection in cattle
Downloaded by [University of South Florida] at 12:22 06 November 2014
B. 0. Fagbemi and I. 0. Obarisiagbonl SUMMARY 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.
INTRODUCTION
In cattle that harbour patent infections of Fasciola, the recovery of eggs from their faeces remains the surest method of diagnosis of liver fluke infection in spite of the vast array of serodiagnostic techniques that have evolved and been described over the past two decades (11). Apart from their inability to detect prepatent infections, the cumbersomeness and the apparent lack of sensitivity of conventional coprological methods coupled with the need for simple, rapid and sensitive diagnostic techniques that are applicable to epidemiological investigations have justified the search for reliable serotests. One serodiagnostic test that has been adapted for the diagnosis of fascioliasis more recently is the enzyme-linked immunosorbent assay (ELISA) (1, 2, 3, 4, 5, 6, 9). In most of these works, ELISA was used for the diagnosis of Fasciola hepatica infection in experimentally infected animals. Furthermore, despite the elaborateness of some of these experimental designs which used different antigen types and protein fractions of E hepatica, there is still a need to truly assess the reliability of ELISA as a diagnostic tool in natural fascioliasis in relation to conventional parasitological techniques especially for E gigantica infection in African cattle. In this study, the sensitivity of ELISA in the diagnosis of naturally occurring E
gigantica infection in cattle was compared to conventional parasitological techniques.
MATERIALS AND METHODS
All the cattle studied were slaughtered at the Ibadan Municipal Abattoir, Nigeria. The age of the animals at the time of slaughter was 2 to 3 years. Sample collection: As described below, samples were collected from four groups of 688 slaughtered cattle: Group I: Blood samples were collected after slaughter from the hearts of 44 cattle whose livers were observed to be infected with E gigantica and from another set of 44 cattle whose livers were fluke-free. I
B. 0. Fagbemi and I. 0. Obarisiagbon, Department of Veterinairy Microbiology and Parasitology, University of Ibadan, lbadan, Nigeria.
THE VETERINARY QUARTERLY, VOL
12, No. I,
JANUARY 1990
35
Downloaded by [University of South Florida] at 12:22 06 November 2014
Group 2: Two hundred blood samples were collected randomly from cattle during slaughtering; their corresponding faecal samples were collected directly from the rectum. Group 3: Two hundred blood samples and faecal samples were also collected randomly from another group of cattle at slaughter. Group 4: In addition to blood and faecal samples, bile samples were randomly collected from the gall bladders of 200 cattle at slaughter irrespective of whether the livers were Fasciola-infected or Fasciola-free. Investigation procedure: ELISA was conducted on all the sera obtained from the four groups of animals. Foetal calf serum was used as Fasciola-free control serum. For the ELISA, E gigantica was prepared by homogenising 20 freshly collected adult flukes in 10 ml of phosphate buffered saline (PBS pH 7.2). The homogenate was left overnight at 4°C before it was centrifuged at 12,000 rpm for one hour at 4°C. The supernatant was stored at 4°C until used. A chequerboard titration (8) was initially performed to determine the optimum antigen concentration for adsorption to the plates and the optimum dilution of both the serum
and the conjugate. The procedure followed for the conduction of the ELISA was a modification of that of Burden and Hammet (2). The antigen was diluted in a carbonate buffer (pH 9.6) to give a solution containing 10
mg/m1; 100 pl of the solution was allowed to adsorb into the wells of polystrene microtitration plates for two hours at 37°C with one minute of shaking at 40-minute
intervals. The wells were washed 12 times with PBS containing 0.05% Tween 20, after which
the serum samples, diluted at a ratio of 1:100 with PBS, were added. The plates were incubated at 37°C for one hour. After washing again as described previously, peroxidaselabelled anti-bovine immunoglobulin (diluted at a ratio of 1:2000 with PBS) was added and the plates were incubated at 37°C for one hour with shaking after 30 minutes. The plates were washed once more and the enzyme substrate orthophenylene diamine, diluted I mg/m1 in 0.05% citrate buffer (pH 5.5), was added. After optimum colour development was allowed for 30 minutes, the reaction was stopped by the addition of 1M sulphuric acid. The optical density was determined spectrophotometrically at 405nm. The tests for all samples were run simultaneously. At the conclusion of the assays, the optical density readings for each group, including that for the negative control, were compared
to detect significant differences between group means. P values less than 0.05 were
considered significant. Particular cut-off points were not used. Parasitological studies: The faecal samples obtained from Group 2 animals were examined for E gigantica eggs by the sedimentation method (10), while those of Group 3 animals
were examined for fluke eggs by the zinc flotation method (7). In Group 4, both the sedimentation and flotation methods were used for the detection of the eggs from each
faecal sample. The bile collected from each animal in this group was examined for Fasciola eggs by sedimentation. For this, 25 ml of bile was centrifuged at 3,000 rpm for 30 minutes and the sediment was examined for Fasciola eggs. Study plan: The study conducted with Group I was designed to compare ELISA results for animals that were observed to be Fasciola-infected and Fasciola-free during post mortem examination. The purpose of the Group 2 studies was to compare the ELISA results with those obtained for the diagnosis of fascioliasis by the sedimentation method, while the purpose of the Group 3 studies was to allow a similar comparison with the flotation
technique. With Group 4, a simultaneous comparison of the four methods (ELISA,
sedimentation, flotation and post mortem diagnosis) was made. In Groups 2 and 3, samples were obtained from different sets of animals to broaden the
sample base and to obtain an independent comparison prior to the simultaneous comparison that was made with Group 4. RESULTS
The ELISA optical density (0D405) readings in the groups of cattle are presented in Tables 1 and 2. In Table 1, the OD readings obtained for cattle with Fasciolainfected livers (0.512 ± 0.181) was significantly higher than those for animals whose livers were Fasciola-free (0.135 ± 0.045). Similarly in Table 1, the ELISA result for sera of animals whose faeces contained 36
THE VF1TRINARY QIJARTFRIN,
Vol. 12, No 11 JArly
Table I. Antibody response to E gigantica infection in three groups of cattle as measured by ELISA (OD 405 nm ).
GROUPS Method of parasitological dia gnosis Liver examination
2 Egg sedimentation
Egg flotation
ELISA OD in sera of cattle positive for Fasciola according to method:
0.512 ± 0,181
0.424 ± 0.031
0.492 ± 0.170
ELISA OD in sera of cattle negative for Fasciola according to method:
0.135 ± 0.045
0.206 ± 0.167
0.416 ± 0.101
Downloaded by [University of South Florida] at 12:22 06 November 2014
1
3
ELISA OD in foetal calf serum = 0.104 ± 0.002
Fasciola eggs as detected by the sedimentation technique (0.424 ± 0.031) was significantly higher than for the cattle without eggs in faeces (0.206 ± 0.167). However, as shown in the same table, the OD values for cattle that were known to be Fasciola-infected as revealed by egg flotation (0.492 ± 0.170) was not significantly different from those of Fasciola-free cattle (0.416 ± 0.101). A simultaneous comparison of ELISA OD values for cattle that were ascertained
to be Fasciola-infected or Fasciola-free by bile egg sedimentation, faecal egg sedimentation and faecal egg flotation is shown in Table 2. It can be seen that while OD values for cattle that were proven to be Fasciola-infected by bile egg sedimentation and faecal egg sedimentation were significantly higher than those for animals that were Fasciola-free, the OD values for the same animals that were categorised by faecal egg flotation were not significantly different from each other. Table 2. Simultaneous comparison of ELISA OD values in a group of cattle in relation to parasitological diagnosis of E gigantica.
Method of parasitogical diagnosis 1
2
3
Bile egg
sedimentation
Faecal egg sedimentation
Faecal egg flotation
ELISA OD in sera of cattle positive for Fasciola according to method:
0.601 ± 0.034
0.462 ± 0.110
0.510 ± 0.061
EL1SA OD in sera of cattle negative for Fasciola according to method:
0.217 ± 0.052
0.214 ± 0.047
0.453 ± 0.059
ELISA OD in foetal calf serum = 0.101 ± 0.005 DISCUSSION
The results from these studies reveal that ELISA results for Fasciola-infected cattle were significantly higher than for the Fasciola-free animals when the methods used for determining the Fasciola-infection was liver examination, bile sedimentation
or faecal egg sedimentation. The difference in OD readings for the Fasciolainfected and the Fasciola-free animals was significant enough to justify positive diagnosis by the serological test. However, reliable results were not obtained when ELISA was used in relation to diagnosis of fascioliasis by faecal egg flotation. Illf tITIOolfor (1111010hr rill (1 it I
I
1
p
)1,
5.
)j.)
l.
,-
timR
11
Downloaded by [University of South Florida] at 12:22 06 November 2014
The equivocal results obtained in this latter case reveal the insensitivity of the faecal flotation method in the diagnosis of bovine fascioliasis. For instance simultaneous comparison of bile sedimentation, faecal egg flotation methods showed that many animals that were classified as Fasciola-free by faecal egg flotation were actually revealed to be Fasciola-infected by the two other methods. It was also observed during this study that zinc sulphate solution, because of its high specific gravity, caused considerable physical distortion of fluke eggs during flotation. Since the main aim of this investigation was to compare ELISA (OD) readings for the different groups 'of animals and not to establish strict positive or negative ELISA values, cut-off values were not set. The present findings confirm the usefulness of ELISA for the diagnosis of natural E gigantica infection as it had been shown for experimental E hepatica infection in cattle (2, 4) and sheep (11). These investigations have not taken into consideration the complications that may be caused by concurrent helminth infections with which E gigantica may share common antigens. In this connection. although Zimmerman et al. (11) demonstrated that concurrent nematode infections did not interfere with ELISA results obtained for experimental ovine fascioliasis, it is possible that antigens shared with more closely related trematodes such as schistosomes may affect the reliability of the test. Furthermore, the present studies have shown that reliable results may be obtained with the use of whole worm extracts as the antigen source. However, in view of the demonstration (9) that fractionated antigens revealed different stages of Fasciola infection in experimental fascioliasis in cattle, the use of fractionated antigens for diagnosing natural fascioliasis in cattle may produce more informative epidemiological data. ACKNOWLEDGEMENTS
This study was supported by the Danish International Development Agency (DANIDA), Denmark. REFERENCES
I. Ambroise-Thomas P, Desgeorges PT, Bouttaz M. Le diagnostic immunoenymologique (ELISA) de la humaine el bovine. Detection d'anticorps ef/ou d'antigenes circulants. Ann Soc Med Trop 1980; 60: 47-60.
2.
Burden DJ, Hammet NC. Microplate enzyme-linked immunosorbent assay for antibody to
Fasciola hepatica in cattle. Vet Rec 1978; 103: 158.
3. Burden DJ, Hammet NC. The ELISA test for detection of Fasciola hepatica infection in cattle. Parasitology 1979; 79: 3.
4.
Farrell CJ, Shen DT, Wescott RB, Lang BZ. An enzyme-linked immunosorbent assay for diagnosis of Fasciola hepatica infection in cattle. Am J Vet Res 1981; 42: 237-40.
Hillyer GV, Santiago de Weil N. Use of immunologic techniques to detect chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme-linked immunosorbent assay in infected rats and rabbits. J Parasitol 197; 65: 680-4. 6. Levine DM, Hillyer GV, Flores SI. Comparison of counterelectrophoresis, the enzyme-linked immunosorbent assay and kato faecal examination for the diagnosis of fascioliasis in infected mice and rabbits. Am J Trop Med Hyg 1980; 29: 602-8. 7. Manual of veterinary parasitological laboratory techniques. London (Technical bulletin n6. 18). Ministry of Agriculture, Fisheries and Food, 1971. 8. McLaren ML, Lillywhite JE, An ACS. Indirect enzyme-linked immunosorbent assay (ELISA): practical aspects of standardieation and quality control. Medical Laboratory Sciences 1981; 38: 5.
245-51.
9. Oldham G. Antibodies to Fasciola hepatica antigens during experimental infections in cattle 10.
measured by ELISA. Vet Parasitol 1983; 13: 151-8. Parfitt JW, Banks AW. A method for counting Fasciola eggs in cattle faeces in the field. Vet Rec 1977; 87: 180-2.
11. Zimmerman GL, HJ en LW, Cerro JE, Farasworth BS, Wescott RB. Diagnosis of Fasciola hepatica infections in sheep by an enzyme-linked immunosorbent assay. Am J Vet Res 1982; 43: 20972100.
38
THE VETERINARY QUARTERLY. VOL. 12, No. 1, JANUARY 1990
