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Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2
Veterinary Parasitology, 45 (1992) 81-88 Elsevier Science Publishers B.V., Amsterdam
81
Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2 Didier Levieuxa, Annie Levieuxa, Christian Mageb and Jean-Pierre Garel c "INRA, Station de Recherches sur la Viande, Unit~ d'Immunochimie, Theix, 63122 St. Genbs-Champanelle, France bNouvel Institut de l'Elevage, Rue Martial Pradet, 87036 Limoges, France ClNRA, D~partement d'Elevage et de Nutrition des Herbivores, Domaine Experimental de Marcenat, 15190 Marcenat, France (Accepted 7 May 1992)
ABSTRACT Levieux, D., Levieux, A., Mage, C. and Garel, J.-P., 1992. Immunologicaldetection of chemotherapeutic success in bovine fasciolosis using the specific antigen t2. Vet. Parasitol., 45:81-88. An improved hemagglutination (HA) test using the purified specific t2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for the prediction of chemotherapeutic success in natural bovine infections with F. hepatica. Lactating cows (n = 16) from a herd naturally infected with F. hepatica were successively treated with nitroxynil (Dovenix, Specia ) and with oxyclozanide (Zanil, ICI) 1 month later. Their 12-specificantibodies were significantly lower than those of a nontreated control group ( n = 15) from the second month after the first treatment, and continued to decline thereafter to negative values 5-6 months post-treatment. In a second experiment, culled and fattened cows (n = 32 ) of unknown fasciolosis history were treated with closantel ( Janssen Pharmaceutica). Three months after treatment, f2-specific antibodies of the serologically positive animals (n=24) were reduced nine-fold. In contrast, in the control group (n=28), the titers of 12-specific antibodies of the serologically positive animals (n = 21 ) were not modified significantly. The results show that the f2-HA test is useful for the prediction of chemotherapeutic success in bovine fascioliasis.
INTRODUCTION
An improved passive hemagglutination (HA) test using the specific f2 antigen of Fasciola hepatica (Biguet et al., 1962; Tailliez et al., 1967) has been recently developed and demonstrated to be more highly specific, simpler, faster and more sensitive than a marketed ELISA kit, on sera from herds with known Correspondence to: D. Levieux, INRA, Station de Recherches sur la Viande, Unit6 d'Immunochimie, Theix, 63122 St. Gen6s-Champanelle, France.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
82
D. LEVIEUX ET AL.
status relating to fasciolosis, or by comparison with the liver infection status of slaughtered animals (Levieux et al., 1992a). Moreover, the cut-off point between populations of negative and positive sera was easy to define, in contrast to the ELISA kit or reported ELISA using semi-purified metabolic/secretory products (Sinclair and Wassal, 1988 ). The developed assay has also been proved particularly suited for the early immunodiagnosis of fasciolosis as f2-specific antibodies were detected 2-4 weeks after infection and persisted throughout the 28 weeks of the experimental period (Levieux et aL, 1992b). Here, we report on the assessment of the improved HA test with regard to its potential use for the prediction of the response to drug therapy of naturally infected cows. Coproscopic analysis is not suitable for this task owing to fecal egg count change during treatment, without killing the worm (Hillyer and Del Llano de Diaz, 1976). MATERIALS AND METHODS
Reagents Bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Sigma (St. Louis, MO ). Mayer's buffer ( 5 mM sodium barbital, 150 mM NaC1, 0.5 mM MgC12, and 0.15 mM CaC12, pH 7.3) was purchased as "complement fixation tablets" from BioLyon (Lyon, France). Mayer-HSA buffer was prepared by adding HSA ( 1 g 1-1 ) and sodium azide ( 1 g 1-1 ) to Mayer's buffer. Glutaraldehyde (25% aqueous solution) was obtained from Serva (Heidelberg, Germany). Other chemicals were supplied by Prolabo (Paris, France). V-bottom microtitration plates were purchased from Greiner (Paris, France).
Sera Blood samples were collected in siliconized tubes by venipuncture and allowed to clot at room temperature. Sera were stored at - 20 ° C until tested.
Antigen f2 Flukes obtained from cattle livers at a slaughterhouse were briefly washed in 0.15 M NaC1 and incubated in 0.015 M NaC1 overnight at 4°C under rotative stirring. The specific antigen f2 of F. hepatica was then purified from the excretory-secretory products essentially as described by Tailliez and Korach (1970a).
DETECTION OF CHEMOTHERAPEUTIC SUCCESS USING ANTIGEN t2
83
Coupling of the f2 antigen to sheep red blood cells (SRBC) Glutaraldehyde has been used as a cross-linking agent as described by Avrameas (1969), with modifications described in detail elsewhere (Levieux et al., 1992a). The SRBC were diluted to 1.2× 108 cells m1-1 in Mayer-HSA buffer (5 mM sodium barbital, 150 mM NaC1, 0.5 mM MgC12, 0.15 M CaC12 and HSA 1 g 1-1, pH 7.3 ) before use.
Passive hemagglutination assay Bovine sera were prediluted at 1/20 dilution in Mayer-HSA buffer containing BSA ( 1 g 1-1 ) and CaC12 (0.05 M). They were titrated using microtiter equipment by double dilution of 0.025 ml of serum in V-bottom plates and adding 0.025 ml of antigen-coated cells. Every sample was checked at the 1/20 dilution with control erythrocytes sensitized with BSA (Levieux et al., 1992a) for the detection of heterophile antibodies. As a positive control, sheep anti-f2 antiserum was tested on each plate. After agitation the microtitration plate was left undisturbed for 1 h at room temperature. The degree of hemagglutination was estimated with the naked eye and recorded as - (0%), + (25%), + + (50%), + + + (75%) or + + + + (100%). Titer was defined as the inverse of the highest dilution showing a hemagglutination, plus the estimated percentage of the hemagglutination at this dilution (i.e. + + at 1/ 4 0 = 4 0 + 4 0 × 5 0 % = 6 0 ; + + + at 1/120= 120+ 120X75%=210). This simple rule allowed more precise quantification than the usual method of recording the highest serum dilution giving complete hemagglutination. In case of positive reaction with the control cells, the heterophile antibodies were absorbed by incubation (30 min, 37°C) of the diluted serum with glutaraldehyde-treated SRBC. All mathematical calculations were made on the log2 of the titer. An arbitrary value of 10 was attributed to titers under the threshold of 20.
Fecal examination Fecal egg counts were made using a sedimentation technique as reported by Raynaud (1970).
Experimental animals In Experiment 1, 31 dairy cows of a moderately infected herd were divided into two groups equilibrated according to serological titers. One group (n = 16) was then treated with nitroxynil (Dovenix, Specia, Paris, France) in the last week of December, which was the end of the dry period. A second treatment was given 6 weeks later in order to kill juvenile flukes not killed by the first
84
D. LEVIEUX ET AL.
treatment. As the cows were at the beginning of lactation, oxyclozanide (Zanil, ICI, Macclesfield, U K ) was used. All treatments were carried out according to the manufacturer's instructions. All the cows were reared in normal farm conditions, housed during winter and reared in infected fields from April. Blood samples were taken monthly until the eighth m o n t h post-treatment. In Experiment 2, 60 culled dairy cows of unknown history with respect to fascioliasis were divided into two groups according to serological and coproscopical results. In one group (n = 32 ), each cow was injected subcutaneously with 20 ml of closantel (Janssen Pharmaceutica, Birse, Belgium). All the animals were housed and fattened on corn silage and soybean meal throughout the 12 weeks of the experimental period. Thereafter they were killed at a local slaughterhouse and the parasite burden in the liver of each cow was determined. The flukes were recovered by dissecting the bile ducts. Then the liver was sectioned into pieces 1-2 cm: in size which were kept in normal saline for 3-4 h and carefully squeezed; the flukes which emerged were collected and counted. The saline was then filtered through a 650 # m pore sieve and the complete flukes or fluke heads recovered were counted. RESULTS
Figure 1 shows the mean and standard error of the results obtained for the first experiment. The pretreatment HA titers for all cows were moderate, indicating a low level of infection. In the control cows, the f2-specific antibodies rose slightly during the first m o n t h post-treatment and stabilized during the second month. Then the antibody level declined slowly and uniformly throughout the remainder of the experimental period, i.e. after the infection 160
T1
T2
+1
== 80
._= < N
4O
.o ._~ "~
20 0
2
4
6
8
Months post treatment Fig. 1. Evolution o f f2-specific antibodies after c h e m o t h e r a p y of Fasciola hepatica-infected lactating dairy cows. T1, t r e a t m e n t with Dovenix; T2, t r e a t m e n t with Zanil. O - O , treated cows (n = 16 ); 0 - O , n o n - t r e a t e d cows ( n = 15 ).
DETECTION OF CHEMOTHERAPEUTIC SUCCESS USING ANTIGEN f2
All animals
85
HA positive animals
2560 -
'=
160
40-
~ 0
12 Weeks
0
12
post-treatment
Fig. 2. Evolution of f2-specific antibodies after chemotherapy of Fasciola hepatica-infected culled dairy cows with closantel. W h i t e bars, n o n - t r e a t e d cows; h a t c h e d bars, treated cows. All animals, treated cows n = 32; n o n - t r e a t e d cows n = 28. H A positive animals, treated cows n = 24; n o n - t r e a t e d cows n = 21.
TABLE 1 Results of coproscopic analysis and of post-mortem examination of livers from culled dairy cows treated or not treated with closantel 12 weeks earlier
Total number of cows
Non-treated
Treated
COWS
COWS
28
32
Total number of cows positive by coproscopic analysis
5 (17.9%)
0 (0%0)
Total number of infected liver
7 (25.0%)
3 (9.4%)
Mean number of flukes recovered per infected liver (mean + SE)
20.8 a +8.5
3.3 a +0.3
aSignificantly different (Mann-Whitney U test, P = 0.026).
had been established in the bile ducts. In contrast, values for the treated cows declined slowly during the first month and then sharply. Because of the high titer variation between animals in each group, differences between control and treated animals were only statistically significant from the second month (Student's t-tests, P<0.01 ). The HA titers were practically negative 5-6 months post-treatment, after which they rose, probably because of a new summer infection.
86
D. LEVIEUX ET AL.
Results of coproscopic analysis were negative throughout the experiment, as expected according to the moderate HA titers. Figure 2 shows the mean and standard error of the results obtained for the second experiment. Twelve weeks post-treatment the HA titers of the control group were not significantly modified. In contrast, the treated group showed a five-fold reduction of HA titers (Student's t-test, P<0.001 ). When only HA-positive animals were considered, results were more demonstrative, showing a nine-fold reduction of HA titers (Student's t-test, P < 0.0001 ). The results of coproscopic analysis and of post-mortem examination at the slaughterhouse are summarized in Table 1. They demonstrated the infection status of the culled cows and the success of the chemotherapy with closantel, as after treatment there were no positive coproscopic results and a significant reduction in the number of flukes recovered from the infected livers in the treated group (Mann-Whitney U-test, P = 0.026 ). DISCUSSION
Numerous studies have evaluated different antigen preparations for the immunodiagnosis of F. hepatica infections but less has been reported on immunological changes following chemotherapy. A reduction in antibodies after anthelminthic treatment has been observed in sheep (Benex et al., 1973 ), rats and rabbits (Hillyer and Del Llano de Diaz, 1976; Hillyer and Allain, 1979) using qualitative precipitation tests such as immunodiffusion, immunoelectrophoresis or counter-electrophoresis, and crude extracts of F. hepatica as test antigens. In contrast, with an HA test using an adult worm homogenate extracted with ether, Hillyer and Allain ( 1979 ) obtained erratic results and were unable to evaluate chemotherapeutic success in infected rabbits. Using a kinetic ELISA, Wyckoff (1983) was also unable to evaluate the anthelminthic efficacy in infected calves and concluded in favor of purifying the antigens used for the diagnosis. Hillyer and Santiago de Weil (1979) developed an ELISA test using extracts of adult worm partially purified using Sephadex G-200 to eliminate the cross-reactivity with antisera containing antibodies to other parasites. However, in both successfully treated rats and rabbits, antibody levels dropped moderately and stabilized afterwards. The difficulty in choosing a satisfactory antigen has been exemplified by Hanna et al. ( 1982 ) who observed that the treatment of sheep 3 or 6 weeks after infection prevented the development of antibodies against T2 (adult tegument) antigen but not against T1 (juvenile tegument glycocalyx) or G (gut secretions) antigens. However, according to the authors, the T2 antibody decline following chemotherapy could not readily be distinguished from natural antibody decline after parasite invasion of the bile ducts, and the indirect immunofluorescent technique used was thus unsuitable to indicate the success of chemotherapy.
DETECTIONOF CHEMOTHERAPEUTICSUCCESSUSINGANTIGENt2
87
In view of the disparity in techniques and test antigens, the results presented here cannot readily be correlated with those of earlier studies. However, they demonstrate that the f2-HA test can indicate clearly the success of chemotherapy shortly after drug administration as in lactating cows a significant (P<0.01) drop in f2-specific antibodies was observed 2 months after treatment and the antibody decline continued thereafter (Fig. 1). Even more demonstrative results were obtained in a second experiment using culled and fattened cows and a different chemotherapeutic scheme, with nine-fold reduction of the HA titers of positive animals observed 3 months after treatment. As the half-lives of bovine IgG1 and IgG2 are respectively 13 days and 20 days (Levieux, 1990), the fast decrease of antibody synthesis together with normal catabolism of IgG could explain the rapid disappearance of f2-specific antibodies. The f2-antigen is one of the major precipitation arcs observed in infected humans or animals using immunoelectrophoresis (Biguet et al., 1962) and this could partly explain our results as positive results of earlier studies had been essentially obtained with precipitation tests. Moreover, our results were obtained on naturally infected animals and not in experimentally infected models as used by earlier workers. In natural infections, the specificity of the antigen is an important prerequisite as antibodies directed against concomitant parasites could cross-react with the test antigen and mask the decline of specific antibodies. In this respect, the high specificity of the f2 antigen which has been previously demonstrated in humans, experimental animals and in bovine species (Biguet et al., 1962; Tailliez et al., 1967; Tailliez and Korach, 1970b; Capron et al., 1975; Levieux et al., 1992a) could contribute to the effectiveness of the f2-HA test for monitoring the success of chemotherapy. REFERENCES Avrameas, S., 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry, 6: 43-52. Benex, J., Guilhon, J. and Barnabe, R., 1973. Etude comparative de diverses m6thodes de diagnostic immunologique de la fasciolose h6pato-biliaire exp6rimentale du mouton et influence du traitement sur la persistance des anticorps. Bull. Soc. Pathol. Exotique, 66:116-118. Biguet, J., Capron, A. and Tran van Ky, P., 1962. Les antig6nes de Fasciola hepatica. Etude 61ectrophor6tique et immuno61ectrophor6tique. Identification des fractions et comparaison avec les antig6nes correspondant ~ sept autres helminthes. Ann. Parasitol., 37:221-231. Capron, A. Bout, D. and Dugimont, J.C., 1975. Apport des m6thodes immunoenzymologiques utilisant des antig6nes purifi6s au diagnostic sp6cifique et automatis6 des infections parasitaires. Ann. Soc. Beige Med. Trop., 55: 443-450. Hanna, R.E.B., Hughes, D.L. and Taylor, S.M., 1982. Fasciola hepatica: antibody levels in sheep serum before and after treatment with anthelmintic. Res. Vet. Sci., 33: 328-332. Hillyer, G.V. and Allain, D., 1979. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. III. Comparison of counterelectrophoresis and indirect hemagglutination in infected rabbits. J. Parasitol., 65: 960-963.
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Hillyer, G.V. and Del Llano de Diaz, A., 1976. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. I. Rabbit infections. Am. J. Trop. Med. Hyg., 25:307-311. Hillyer, G.V. and Santiago de Weil, N., 1979. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme linked immunosorbent assay in infected rats and rabbits. J. Parasitol., 65: 680-684. Levieux, D., 1990. lmmunoglobulines et transmission de l'immunit6 passive chez les ruminants. In: P.P. Pastoret, A., Govaerts and H. Bazin (Editors), Immunologie Animale. Flammarion M~decine-Sciences, Paris, pp. 596-599. Levieux, D., Levieux, A. and Venien, A., 1992a. An i.mproved passive hemagglutination test for the serological diagnosis of bovine fascioliasis using the specific antigen f2. Vet. Parasitol., 42: 53-66. Levieux, D., Levieux, A., Mage, C. and Venien, A., 1992b. Early immunodiagnosis of bovine fascioliasis using the specific antigen f2. Vet. Parasitol., in press. Raynaud, J.P., 1970. Etude de l'efficacit6 d'une technique de coproscopie quantitative pour le diagnostic de routine et le contr61e des infestations parasitaires des bovins, ovins, 6quins et porcins. Ann. Parasitol. Hum. Comp., 45: 321-343. Sinclair, I.J. and Wassal, D.A., 1988. Serodiagnosis of Fasciola hepatica infections in cattle. Vet. Parasitol., 27: 283-290. Tailliez, R. and Korach, S., 1970a. Les antig~nes de Fasciola hepatica. I. Isolement et caract6risation d'un antig6ne sp6cifique du genre. Ann. Inst. Pasteur, 118: 61-78. Tailliez, R. and Korach, S., 1970b. Les antig6nes de Fasciola hepatica. II. Etude immunologique et localisation in situ d'un antigone sp6cifique du genre. Ann. Inst. Pasteur, 118: 330-339. Tailliez, R., Mangalo, R. and Korach, S., 1967. Isolement d'un antigone sp6cifique de la grande douve du foie (Fasciola hepatica). C.R. Acad. Sci., 265: 466-469. Wyckoff, J.H., 1983. Diagnosis of Fasciola hepatica infection in beef calves by analysis of plasma constituents. Diss. Abstr. Intern., 43: 2755-2756.
81
Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2 Didier Levieuxa, Annie Levieuxa, Christian Mageb and Jean-Pierre Garel c "INRA, Station de Recherches sur la Viande, Unit~ d'Immunochimie, Theix, 63122 St. Genbs-Champanelle, France bNouvel Institut de l'Elevage, Rue Martial Pradet, 87036 Limoges, France ClNRA, D~partement d'Elevage et de Nutrition des Herbivores, Domaine Experimental de Marcenat, 15190 Marcenat, France (Accepted 7 May 1992)
ABSTRACT Levieux, D., Levieux, A., Mage, C. and Garel, J.-P., 1992. Immunologicaldetection of chemotherapeutic success in bovine fasciolosis using the specific antigen t2. Vet. Parasitol., 45:81-88. An improved hemagglutination (HA) test using the purified specific t2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for the prediction of chemotherapeutic success in natural bovine infections with F. hepatica. Lactating cows (n = 16) from a herd naturally infected with F. hepatica were successively treated with nitroxynil (Dovenix, Specia ) and with oxyclozanide (Zanil, ICI) 1 month later. Their 12-specificantibodies were significantly lower than those of a nontreated control group ( n = 15) from the second month after the first treatment, and continued to decline thereafter to negative values 5-6 months post-treatment. In a second experiment, culled and fattened cows (n = 32 ) of unknown fasciolosis history were treated with closantel ( Janssen Pharmaceutica). Three months after treatment, f2-specific antibodies of the serologically positive animals (n=24) were reduced nine-fold. In contrast, in the control group (n=28), the titers of 12-specific antibodies of the serologically positive animals (n = 21 ) were not modified significantly. The results show that the f2-HA test is useful for the prediction of chemotherapeutic success in bovine fascioliasis.
INTRODUCTION
An improved passive hemagglutination (HA) test using the specific f2 antigen of Fasciola hepatica (Biguet et al., 1962; Tailliez et al., 1967) has been recently developed and demonstrated to be more highly specific, simpler, faster and more sensitive than a marketed ELISA kit, on sera from herds with known Correspondence to: D. Levieux, INRA, Station de Recherches sur la Viande, Unit6 d'Immunochimie, Theix, 63122 St. Gen6s-Champanelle, France.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
82
D. LEVIEUX ET AL.
status relating to fasciolosis, or by comparison with the liver infection status of slaughtered animals (Levieux et al., 1992a). Moreover, the cut-off point between populations of negative and positive sera was easy to define, in contrast to the ELISA kit or reported ELISA using semi-purified metabolic/secretory products (Sinclair and Wassal, 1988 ). The developed assay has also been proved particularly suited for the early immunodiagnosis of fasciolosis as f2-specific antibodies were detected 2-4 weeks after infection and persisted throughout the 28 weeks of the experimental period (Levieux et aL, 1992b). Here, we report on the assessment of the improved HA test with regard to its potential use for the prediction of the response to drug therapy of naturally infected cows. Coproscopic analysis is not suitable for this task owing to fecal egg count change during treatment, without killing the worm (Hillyer and Del Llano de Diaz, 1976). MATERIALS AND METHODS
Reagents Bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Sigma (St. Louis, MO ). Mayer's buffer ( 5 mM sodium barbital, 150 mM NaC1, 0.5 mM MgC12, and 0.15 mM CaC12, pH 7.3) was purchased as "complement fixation tablets" from BioLyon (Lyon, France). Mayer-HSA buffer was prepared by adding HSA ( 1 g 1-1 ) and sodium azide ( 1 g 1-1 ) to Mayer's buffer. Glutaraldehyde (25% aqueous solution) was obtained from Serva (Heidelberg, Germany). Other chemicals were supplied by Prolabo (Paris, France). V-bottom microtitration plates were purchased from Greiner (Paris, France).
Sera Blood samples were collected in siliconized tubes by venipuncture and allowed to clot at room temperature. Sera were stored at - 20 ° C until tested.
Antigen f2 Flukes obtained from cattle livers at a slaughterhouse were briefly washed in 0.15 M NaC1 and incubated in 0.015 M NaC1 overnight at 4°C under rotative stirring. The specific antigen f2 of F. hepatica was then purified from the excretory-secretory products essentially as described by Tailliez and Korach (1970a).
DETECTION OF CHEMOTHERAPEUTIC SUCCESS USING ANTIGEN t2
83
Coupling of the f2 antigen to sheep red blood cells (SRBC) Glutaraldehyde has been used as a cross-linking agent as described by Avrameas (1969), with modifications described in detail elsewhere (Levieux et al., 1992a). The SRBC were diluted to 1.2× 108 cells m1-1 in Mayer-HSA buffer (5 mM sodium barbital, 150 mM NaC1, 0.5 mM MgC12, 0.15 M CaC12 and HSA 1 g 1-1, pH 7.3 ) before use.
Passive hemagglutination assay Bovine sera were prediluted at 1/20 dilution in Mayer-HSA buffer containing BSA ( 1 g 1-1 ) and CaC12 (0.05 M). They were titrated using microtiter equipment by double dilution of 0.025 ml of serum in V-bottom plates and adding 0.025 ml of antigen-coated cells. Every sample was checked at the 1/20 dilution with control erythrocytes sensitized with BSA (Levieux et al., 1992a) for the detection of heterophile antibodies. As a positive control, sheep anti-f2 antiserum was tested on each plate. After agitation the microtitration plate was left undisturbed for 1 h at room temperature. The degree of hemagglutination was estimated with the naked eye and recorded as - (0%), + (25%), + + (50%), + + + (75%) or + + + + (100%). Titer was defined as the inverse of the highest dilution showing a hemagglutination, plus the estimated percentage of the hemagglutination at this dilution (i.e. + + at 1/ 4 0 = 4 0 + 4 0 × 5 0 % = 6 0 ; + + + at 1/120= 120+ 120X75%=210). This simple rule allowed more precise quantification than the usual method of recording the highest serum dilution giving complete hemagglutination. In case of positive reaction with the control cells, the heterophile antibodies were absorbed by incubation (30 min, 37°C) of the diluted serum with glutaraldehyde-treated SRBC. All mathematical calculations were made on the log2 of the titer. An arbitrary value of 10 was attributed to titers under the threshold of 20.
Fecal examination Fecal egg counts were made using a sedimentation technique as reported by Raynaud (1970).
Experimental animals In Experiment 1, 31 dairy cows of a moderately infected herd were divided into two groups equilibrated according to serological titers. One group (n = 16) was then treated with nitroxynil (Dovenix, Specia, Paris, France) in the last week of December, which was the end of the dry period. A second treatment was given 6 weeks later in order to kill juvenile flukes not killed by the first
84
D. LEVIEUX ET AL.
treatment. As the cows were at the beginning of lactation, oxyclozanide (Zanil, ICI, Macclesfield, U K ) was used. All treatments were carried out according to the manufacturer's instructions. All the cows were reared in normal farm conditions, housed during winter and reared in infected fields from April. Blood samples were taken monthly until the eighth m o n t h post-treatment. In Experiment 2, 60 culled dairy cows of unknown history with respect to fascioliasis were divided into two groups according to serological and coproscopical results. In one group (n = 32 ), each cow was injected subcutaneously with 20 ml of closantel (Janssen Pharmaceutica, Birse, Belgium). All the animals were housed and fattened on corn silage and soybean meal throughout the 12 weeks of the experimental period. Thereafter they were killed at a local slaughterhouse and the parasite burden in the liver of each cow was determined. The flukes were recovered by dissecting the bile ducts. Then the liver was sectioned into pieces 1-2 cm: in size which were kept in normal saline for 3-4 h and carefully squeezed; the flukes which emerged were collected and counted. The saline was then filtered through a 650 # m pore sieve and the complete flukes or fluke heads recovered were counted. RESULTS
Figure 1 shows the mean and standard error of the results obtained for the first experiment. The pretreatment HA titers for all cows were moderate, indicating a low level of infection. In the control cows, the f2-specific antibodies rose slightly during the first m o n t h post-treatment and stabilized during the second month. Then the antibody level declined slowly and uniformly throughout the remainder of the experimental period, i.e. after the infection 160
T1
T2
+1
== 80
._= < N
4O
.o ._~ "~
20 0
2
4
6
8
Months post treatment Fig. 1. Evolution o f f2-specific antibodies after c h e m o t h e r a p y of Fasciola hepatica-infected lactating dairy cows. T1, t r e a t m e n t with Dovenix; T2, t r e a t m e n t with Zanil. O - O , treated cows (n = 16 ); 0 - O , n o n - t r e a t e d cows ( n = 15 ).
DETECTION OF CHEMOTHERAPEUTIC SUCCESS USING ANTIGEN f2
All animals
85
HA positive animals
2560 -
'=
160
40-
~ 0
12 Weeks
0
12
post-treatment
Fig. 2. Evolution of f2-specific antibodies after chemotherapy of Fasciola hepatica-infected culled dairy cows with closantel. W h i t e bars, n o n - t r e a t e d cows; h a t c h e d bars, treated cows. All animals, treated cows n = 32; n o n - t r e a t e d cows n = 28. H A positive animals, treated cows n = 24; n o n - t r e a t e d cows n = 21.
TABLE 1 Results of coproscopic analysis and of post-mortem examination of livers from culled dairy cows treated or not treated with closantel 12 weeks earlier
Total number of cows
Non-treated
Treated
COWS
COWS
28
32
Total number of cows positive by coproscopic analysis
5 (17.9%)
0 (0%0)
Total number of infected liver
7 (25.0%)
3 (9.4%)
Mean number of flukes recovered per infected liver (mean + SE)
20.8 a +8.5
3.3 a +0.3
aSignificantly different (Mann-Whitney U test, P = 0.026).
had been established in the bile ducts. In contrast, values for the treated cows declined slowly during the first month and then sharply. Because of the high titer variation between animals in each group, differences between control and treated animals were only statistically significant from the second month (Student's t-tests, P<0.01 ). The HA titers were practically negative 5-6 months post-treatment, after which they rose, probably because of a new summer infection.
86
D. LEVIEUX ET AL.
Results of coproscopic analysis were negative throughout the experiment, as expected according to the moderate HA titers. Figure 2 shows the mean and standard error of the results obtained for the second experiment. Twelve weeks post-treatment the HA titers of the control group were not significantly modified. In contrast, the treated group showed a five-fold reduction of HA titers (Student's t-test, P<0.001 ). When only HA-positive animals were considered, results were more demonstrative, showing a nine-fold reduction of HA titers (Student's t-test, P < 0.0001 ). The results of coproscopic analysis and of post-mortem examination at the slaughterhouse are summarized in Table 1. They demonstrated the infection status of the culled cows and the success of the chemotherapy with closantel, as after treatment there were no positive coproscopic results and a significant reduction in the number of flukes recovered from the infected livers in the treated group (Mann-Whitney U-test, P = 0.026 ). DISCUSSION
Numerous studies have evaluated different antigen preparations for the immunodiagnosis of F. hepatica infections but less has been reported on immunological changes following chemotherapy. A reduction in antibodies after anthelminthic treatment has been observed in sheep (Benex et al., 1973 ), rats and rabbits (Hillyer and Del Llano de Diaz, 1976; Hillyer and Allain, 1979) using qualitative precipitation tests such as immunodiffusion, immunoelectrophoresis or counter-electrophoresis, and crude extracts of F. hepatica as test antigens. In contrast, with an HA test using an adult worm homogenate extracted with ether, Hillyer and Allain ( 1979 ) obtained erratic results and were unable to evaluate chemotherapeutic success in infected rabbits. Using a kinetic ELISA, Wyckoff (1983) was also unable to evaluate the anthelminthic efficacy in infected calves and concluded in favor of purifying the antigens used for the diagnosis. Hillyer and Santiago de Weil (1979) developed an ELISA test using extracts of adult worm partially purified using Sephadex G-200 to eliminate the cross-reactivity with antisera containing antibodies to other parasites. However, in both successfully treated rats and rabbits, antibody levels dropped moderately and stabilized afterwards. The difficulty in choosing a satisfactory antigen has been exemplified by Hanna et al. ( 1982 ) who observed that the treatment of sheep 3 or 6 weeks after infection prevented the development of antibodies against T2 (adult tegument) antigen but not against T1 (juvenile tegument glycocalyx) or G (gut secretions) antigens. However, according to the authors, the T2 antibody decline following chemotherapy could not readily be distinguished from natural antibody decline after parasite invasion of the bile ducts, and the indirect immunofluorescent technique used was thus unsuitable to indicate the success of chemotherapy.
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In view of the disparity in techniques and test antigens, the results presented here cannot readily be correlated with those of earlier studies. However, they demonstrate that the f2-HA test can indicate clearly the success of chemotherapy shortly after drug administration as in lactating cows a significant (P<0.01) drop in f2-specific antibodies was observed 2 months after treatment and the antibody decline continued thereafter (Fig. 1). Even more demonstrative results were obtained in a second experiment using culled and fattened cows and a different chemotherapeutic scheme, with nine-fold reduction of the HA titers of positive animals observed 3 months after treatment. As the half-lives of bovine IgG1 and IgG2 are respectively 13 days and 20 days (Levieux, 1990), the fast decrease of antibody synthesis together with normal catabolism of IgG could explain the rapid disappearance of f2-specific antibodies. The f2-antigen is one of the major precipitation arcs observed in infected humans or animals using immunoelectrophoresis (Biguet et al., 1962) and this could partly explain our results as positive results of earlier studies had been essentially obtained with precipitation tests. Moreover, our results were obtained on naturally infected animals and not in experimentally infected models as used by earlier workers. In natural infections, the specificity of the antigen is an important prerequisite as antibodies directed against concomitant parasites could cross-react with the test antigen and mask the decline of specific antibodies. In this respect, the high specificity of the f2 antigen which has been previously demonstrated in humans, experimental animals and in bovine species (Biguet et al., 1962; Tailliez et al., 1967; Tailliez and Korach, 1970b; Capron et al., 1975; Levieux et al., 1992a) could contribute to the effectiveness of the f2-HA test for monitoring the success of chemotherapy. REFERENCES Avrameas, S., 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry, 6: 43-52. Benex, J., Guilhon, J. and Barnabe, R., 1973. Etude comparative de diverses m6thodes de diagnostic immunologique de la fasciolose h6pato-biliaire exp6rimentale du mouton et influence du traitement sur la persistance des anticorps. Bull. Soc. Pathol. Exotique, 66:116-118. Biguet, J., Capron, A. and Tran van Ky, P., 1962. Les antig6nes de Fasciola hepatica. Etude 61ectrophor6tique et immuno61ectrophor6tique. Identification des fractions et comparaison avec les antig6nes correspondant ~ sept autres helminthes. Ann. Parasitol., 37:221-231. Capron, A. Bout, D. and Dugimont, J.C., 1975. Apport des m6thodes immunoenzymologiques utilisant des antig6nes purifi6s au diagnostic sp6cifique et automatis6 des infections parasitaires. Ann. Soc. Beige Med. Trop., 55: 443-450. Hanna, R.E.B., Hughes, D.L. and Taylor, S.M., 1982. Fasciola hepatica: antibody levels in sheep serum before and after treatment with anthelmintic. Res. Vet. Sci., 33: 328-332. Hillyer, G.V. and Allain, D., 1979. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. III. Comparison of counterelectrophoresis and indirect hemagglutination in infected rabbits. J. Parasitol., 65: 960-963.
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Hillyer, G.V. and Del Llano de Diaz, A., 1976. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. I. Rabbit infections. Am. J. Trop. Med. Hyg., 25:307-311. Hillyer, G.V. and Santiago de Weil, N., 1979. Use of immunological techniques to detect chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme linked immunosorbent assay in infected rats and rabbits. J. Parasitol., 65: 680-684. Levieux, D., 1990. lmmunoglobulines et transmission de l'immunit6 passive chez les ruminants. In: P.P. Pastoret, A., Govaerts and H. Bazin (Editors), Immunologie Animale. Flammarion M~decine-Sciences, Paris, pp. 596-599. Levieux, D., Levieux, A. and Venien, A., 1992a. An i.mproved passive hemagglutination test for the serological diagnosis of bovine fascioliasis using the specific antigen f2. Vet. Parasitol., 42: 53-66. Levieux, D., Levieux, A., Mage, C. and Venien, A., 1992b. Early immunodiagnosis of bovine fascioliasis using the specific antigen f2. Vet. Parasitol., in press. Raynaud, J.P., 1970. Etude de l'efficacit6 d'une technique de coproscopie quantitative pour le diagnostic de routine et le contr61e des infestations parasitaires des bovins, ovins, 6quins et porcins. Ann. Parasitol. Hum. Comp., 45: 321-343. Sinclair, I.J. and Wassal, D.A., 1988. Serodiagnosis of Fasciola hepatica infections in cattle. Vet. Parasitol., 27: 283-290. Tailliez, R. and Korach, S., 1970a. Les antig~nes de Fasciola hepatica. I. Isolement et caract6risation d'un antig6ne sp6cifique du genre. Ann. Inst. Pasteur, 118: 61-78. Tailliez, R. and Korach, S., 1970b. Les antig6nes de Fasciola hepatica. II. Etude immunologique et localisation in situ d'un antigone sp6cifique du genre. Ann. Inst. Pasteur, 118: 330-339. Tailliez, R., Mangalo, R. and Korach, S., 1967. Isolement d'un antigone sp6cifique de la grande douve du foie (Fasciola hepatica). C.R. Acad. Sci., 265: 466-469. Wyckoff, J.H., 1983. Diagnosis of Fasciola hepatica infection in beef calves by analysis of plasma constituents. Diss. Abstr. Intern., 43: 2755-2756.