This article was downloaded by: [UOV University of Oviedo] On: 10 November 2014, At: 05:47 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20

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

a

J.B. W. J. Cornelissen , W. A. de Leeuw & P. J. van der Heijden

a

a

Department of Immunology , Central Veterinary Institute , P.O. Box 65, Lelystad, 8200 AB, the Netherlands Published online: 01 Nov 2011.

To cite this article: J.B. W. J. Cornelissen , W. A. de Leeuw & P. J. van der Heijden (1992) comparison of an indirect haemagglutination assay and an Elisa for diagnosing Fasciola hepatica in experimentally and naturally infected sheep, Veterinary Quarterly, 14:4, 152-156, DOI: 10.1080/01652176.1992.9694354 To link to this article: http://dx.doi.org/10.1080/01652176.1992.9694354

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, sub-licensing, 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

COMPARISON OF AN INDIRECT HAEMAGGLUTINATION ASSAY AND AN ELISA FOR DIAGNOSING FASCIOLA HEPATICA IN EXPERIMENTALLY AND NATURALLY INFECTED SHEEP J. B. W. J. Cornelissen, W. A. de Leeuw and P. J. van der Heijden1

Downloaded by [UOV University of Oviedo] at 05:47 10 November 2014

Veterinary Quarterly 1992; 14: 152 - 6

SUMMARY An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an

with the IHA results by testing sera from experimentally and naturally infected sheep.

indirect haemagglutination assay (IHA) for ability to detect

MATERIALS AND METHODS

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 respecti-

Animals Ewes of the Texel breed between 3-12 month of age were used in the study. The experimentally infected sheep were reared free of parasites until use.

vely. 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.

Experimental infection with F.hepatica Sheep (n=32) were infected once with 20 metacercariae of Ehepatica and were killed 20 weeks post infection. Serum samples were taken at weekly intervals. Sheep were monitored for infection by weekly counting of the number of eggs in the faeces (3). At slaughter flukes were detected in the bile duct of all sheep.

Infection with other parasites INTRODUCTION

Various antisera against other parasites were used to evaluate the

Fasciola hepatica (E hepatica) can infect a wide range of

specificity of the ELISA. All sheep were infected experimentally, except for the sheep that were naturally infected with

vertebrate hosts, but is found mainly in domestic ruminants such

as cattle, sheep and goats. In these animals E hepatica causes major economic losses throughout the world (5). Diagnosis of Ehepatica infections is usually based on faecal egg counts or serological assays. Several immunoassays for the detection of Ehepatica infections in sheep have been described, including the ELISA (7, 11), the dot-ELISA (12) and the thin-layer immunoassay (6). However, the indirect haemagglutinating assay (IHA) is still used to detect antibodies in sera from suspected.sheep in the Netherlands (8). This technique is not very sensitive and can give rise to false-positive reactions (8). For practical reasons, the ELISA would be the method of choice to replace the IHA in the Netherlands:Most of the ELISAs described use somatic (S) or excretory-secretory (ES) antigens, although some authors have used partionally fractionated antigen preparations. For instance, antigens have been fractionated by affinity chromatography (9), by preparative iso-electrofocussing (10) or by gel chromatogra-

phy (2). However, none of these authors concluded that fractionated antigens improved the sensitivity or specificity of the ELISA compared with unfractionated S or ES antigens. It is therefore not clear whether S or ES antigens are the most appropiate for use in an ELISA. In this study we evaluated the indirect ELISA, as described by Santiago and Hillyer (7), for use in the serodiagnosis of ovine fasciolosis in the Netherlands. We

compared the ELISA results obtained with S or ES antigens

Taenia ovis (Tovis). All sheep infected with other parasites were E hepatica free and mono-infected with the specific parasites as far we could determine. Antisera against Haemonchus contortus orginated from sheep (n=70) infected repeatedly (5-50 times) with doses of 5,000 to 20,000 larvae. Antisera against Osterta-

gia circumcincta orginated from sheep (n=16) infected once with 30,000 larvae. Antisera against Tovis orginated from sheep

(n=8) that had grazed on a pasture contaminated with Tovis eggs. Cysticerci were found in all sheep at slaughter. Antisera against Eimeria spp. orginated from sheep (n=24) infected twice with 10,000 oocysts. Antisera against Trichostrongylus vitrinus, Cooperia curticei and Trichostrongylus colubriformis orginated from sheep (n= 34 16, and 3, respectively) infected once with 20,000 larvae. Antisera against Nematodirus battus orginated from sheep (n=3) infected five times with 5,000 larvae. Blood samples were taken 10-15 weeks after infection, when all infected sheep shed parasite eggs or oocysts. Negative control sera were collected from parasite-free sheep (n=120).

Natural infection with F.hepatica Five groups of eight 3- to 6-month-old lambs of the Texel breed

(n=157) were turned out each year on potentially naturally infected pastures. Each lamb was allowed to graze a surface of approx. 200 m2 and was moved weekly to another place over a

5-week period. After 5 weeks the lambs were moved to a '

Central Veterinary Institute, Department of Immunology, P.O. Box 65, 8200 AB. Lelystad, the Netherlands

1 52

TFi l

VI:

metacercariae-free pasture. The animals were slaughtered 17 weeks after the stars of the experiment. Serum samples were

T E R I N A R Y Q U A R T E R L Y . V O I.

1

4.

No. 4.

DCEMBER

.

1

992

taken at weekly intervals. Sheep were monitored for infection by weekly counting of the number of eggs in the faeces (3). Flukes were collected at slaughter, using a previously described technique (4).

The average protein concentration of the supernatant was 25 pg/ml. The total protein yield was 2.5 mg per 10 g flukes.

Ehepatica antigens Somatic antigens (S) Adult flukes (10 g), collected from the bile ducts of experimentally infected calves, were washed 3-4 times at room tempera-

as described (7). The wells of ELISA plates (Greiner nr. 655001, Alphen aan de Rijn, the Netherlands) were coated with 100 pl

ELISA The indirect ELISA was performed, with slight modifications,

Ehepatica antigen (1.5 pg protein/ml) in 0.05 M carbonate buffer (pH 9.6). The plates were incubated overnight at 37°C and then washed three times with 0.05% Tween-80 in water.

ture for 1 h in 0.01 M phosphate-buffered saline (PBS) (pH 7.0). The flukes were homogenized in an omnimixer (Sorvall) for 10 x 0.5 min at 4 °C and then ultrasonicated three times for 60 s in a sonifier (Marius) at an amplitude of 5. The ground flukes were

The wells were filled with 100 pl serum diluted 1:100 in ELISA buffer (0.05% Tween-80 and 0.5M NaC1 in 0.01 M phosphate buffer (pH 7.5) with 2.5 g milk powder (ABE Kamel, no.9378 Hope farms B.V., Woerden, the Netherlands) per liter ELISA buffer. Two-fold serial dilutions of sera were tested, starting at 1:25 (Figure 1). The plates were incubated for 1 h at 37°C and washed as described. 'Then each well was filled with 100 pl of rabbit anti-sheep immunoglobulins conjugated with horseradish peroxidase (Dakopatts a/s Denmark, P163) diluted 1:30,000 in

Downloaded by [UOV University of Oviedo] at 05:47 10 November 2014

extracted overnight in 100 ml of a mixture of phosphatebuffered saline with 100 U/ml Na penicillin, 0.25 mg/ml streptomycin, 100 U/ml nystatin, 0.05 pg/ml N-Tosyr-Lphenyl-alanylchlormethan, 0.025 pg/ml N-Tosyl L-Lysylchlormethanhydrochlorid and 0.5% (v/v) NP-40 at 4°C. The suspension was centrifuged at 10,000 x g for 20 min at 4°C. The supernatant was filtered (0.20 pm, Aerodisc) and stored at -70 °C until used. The amount of protein was measured by the method of Bradford (1). The average protein concentration of the supernatant was 1 mg/ml. Ten grams flukes yielded 100 mg protein.

ELISA buffer. The plates were incubated for 1 h at 37°C and washed as described. Tetramethylbenzidine (1 mg/ml) and H202 (0.005%) in 0.1M Na-acetate and 0.1M citric acid buffer (pH 6.0) were used to produce a coloured substrate. Thirty minutes after addition of the substrate solution, the wells of the ELISA plates were filled with 0.5M H2SO4, and the extinction

Excretory-secretory antigens (ES) Adult flukes, collected from the bile ducts of experimentally

values were measured at 450 nm in an Easyreader spectrophoto-

infected cows, were washed 3-4 times at room temperature for 1 h with 0.01 M PBS (pH 7.0). The flukes were then incubated (20

calculated as the average plus three times the standard deviation

meter (SLT, Vienne). The cut-off value of the ELISA was of the OD 450 nm of sera from parasite-free sheep at 1:100 dilution (Figure 3, group 10).

worms/1000 ml) in RPM! 1640 containing streptomycin (100

pg/m1) and penicillin (100 IU/ml) at 37°C for 6 days. The medium was refreshed each day. The supernatant collected on day 3 to 6 was pooled and centrifuged at 4°C at 10,000g for 1 h to remove particulate material and stored at -70°C until used.

Indirect haemagglutination assay (IHA) The IHA titre was determined by using sheep red blood cells coated with S antigens according to the method described by

3200

3200

800

800

ELISA-titre

IHA-titre

\ 200

200

50

50

0 0

1

2

I

I

3

4

1

1

6

1

1

8

1

1

1

12

1

1

1

1

1

0

20

16

Weeks after infection 0 ES antigen

S antigen

A

IHA

Figure 1. Average titres measured by ELISA with S antigens or ES antigens and IHA in sera from sheep (n=8) experimentally infected with F hepatica. The S.E. is shown in bars.

1 53

T

R

I

N A R 1( Q U A

IL

FER

I

.

VOI

1

4.

No

4

.

D EcE

I It

.

1

992

specificity of the ELISA with ES antigens was 95% (11 false positive results). The OD 450 nm ELISA value for the control sheep when using S antigens was lower (0.109 ± 0.071) than that when using ES antigens (0.149 ± 0.018). Sera obtained from sheep infected with parasites other than E hepatica were also tested in the IHA (results not shown). The specificity of the IHA was calculated to be 86%.

van Tiggele and Over (8). Sera with a titre of 1:50 or more in the IHA were scored positive. RESULTS

Experimental infection with F.hepatica. Figure 1 shows that 2 weeks post-infection the ELISAs, with S antigens or ES antigens gave signals significantly (p