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Detection of antibodies to Fasciola hepatica excretory-secretory antigens in experimentally infected goats by enzyme immunosorbent assay
veterinary parasitology ELSEVIER
Veterinary Parasitology 62 (I 996) 247-252
Detection of antibodies to Fasciola hepatica excretory-secretory antigens in experimentally infected goats by enzyme immunosorbent assay A. Martfnez *, M.S. Marffnez-Cruz, F.J. Marffnez, P.N. Gutierrez, S. Hemg.ndez Cdtedra de Parasitologlay Enfermedades Parasitarias, Departamentode Sanidad Animal, Facultad de Veterinaria, Universidad de Ctrdoba, 14071 Ctrdoba, EspaYza Received 12 April 1995; accepted 10 July 1995
Abstract An EL1SA with excretory-secretory (ES) antigens has been evaluated as a technique for the early detection of specific antibodies in Fasciola hepatica infections in goats. Goats were experimentally infected with 100 or 200 metacercariae of bovine origin and serum samples were taken periodically over 365 days. The ELISA test was performed with ES antigens (10/xg ml-t), a single dilution of sera (1:800) and anti-goat lgG conjugate (1:1000). ES specific antigens were detected in all infected goats between 15 and 30 days postinfection (PI) and maximum antibody levels were reached at 90 days PI. Positive antibody levels (significantly different from those of controls) were still found at 365 days PI. No significant differences were observed between goats infected with 100 or 200 metacereariae. In all infected goats, eggs appeared in faeces between 60 and 90 days PI. ELISA with ES antigens could be a feasible method for the early diagnosis of goat fasciolosis. Keywords: Goat; Fasciola hepatica; Diagnosis-Trematoda; ELISA
1. I n t r o d u c t i o n Fasciolosis in goats is considered to be a less frequent and less important infection than in cattle and sheep in Europe. However, it is a common infection in many areas of the world: prevalences between 72% in China (Wang et al., 1987), 14% in India
*Corresponding author. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 8 7 6 - 4
248
A. Martinez et al. / Veterinary Parasitology 62 (1996) 247-252
(Sharma et al., 1989) 20% in Chile (Schenone and Rojas, 1988) 18% in Turkey (Toparlak and Giill, 1988) and 20% in Morocco (Khalaayoune and E1-Hari, 1991) have been already reported. Goats have been described as extremely sensitive to natural and experimental infections (Reddington et al., 1986). The disease usually appears in a chronic form but acute fasciolosis with high rates of mortality has been described (Leathers et ai., 1982). As treatment in the initial stages of the infection considerably reduces liver injury, it is therefore desirable to have a simple, sensitive and specific test for the early diagnosis of fasciolosis in goats. Although several immunological tests have been developed for the detection of specific antibodies to F. hepatica infections in cattle and sheep, few of them have been evaluated in goats: only Levieux and Levieux (1994) reported a haemagluttination (HA) test for the early diagnosis of caprine fasciolosis. The ELISA is the most widely used test, because of its simplicity, reliability and easy mechanization, but it has not been used in goats. The ELISA for cattle and sheep has been proved to be sensitive, specific, rapid and easy to perform (Zimmerman et al., 1982). Improved sensitivity and specificity has been obtained with the purification of somatic antigens and the use of excretory-secretory (ES) products of F. hepatica as antigen (Santiago and Hillyer, 1988). The aim of our work was to evaluate the ELISA with ES antigens as a technique for the early detection of specific antibodies to F. hepatica infections in goats, evaluating the dynamics of antibody production throughout the infection and its correlation with faecal egg output. We have used primary experimental infections with two different infective doses of metacercariae and followed the goats for 1 year.
2. Materials and methods
Eighteen "Serrana" breed males, aged 6 months were used in the study. They were left for 3 months to acclimatize to the experimental conditions. The animals were examined to exclude the possibility of any previous F. hepatica infection by coprological and serological methods. They were kept in stalls and fed with hay and a commercial pelleted ration containing 16.5% proteins. Water was available ad libitum. F. hepatica metacercariae were obtained from Lymnea truncatula snails infected with eggs collected from naturally infected cattle. The metacercariae were kept at 4°C for 6 months until used. The goats were divided in three groups: Group 1 consisted of 6 animals infected with 200 metacercariae; Group 2, of 6 animals infected with 100 metacercariae and Group 3, of 6 animals left as uninfected controls. The metacercariae were administered orally in a 5 ml saline suspension. Blood and faecal samples were taken from each animal on Days 0, 15, 30, 45, 60, 75, 90, 105, 125, 145, 195, 245, 305 and 365 pos infection (PI). Serum was separated and stored at - 8 0 ° C until used. Faecal examinations were performed by a sedimentation method (Thienpont et al., 1986)
A. Martfnez et al. / Veterinary Parasitology 62 (1996) 247-252
249
The F. hepatica ES antigen was obtained as described by Poitou et al. (1992). Briefly, adult liver flukes obtained from cattle at the slaughterhouse were washed and finally incubated in physiological saline (pH 7.2) at 37°C for 24 h. The solution was centrifuged at 4°C and 20000 rpm for 30 min to remove eggs and particulate material. The supernatant containing excretory-secretory products (ES) was collected and its protein concentration was measured as described by Lowry et al. (1951). The ELISA was performed in 96 wells microtiter plates (Maxisorp, Nunc). The optimum antigen, serum and conjugate concentrations and the incubation times were previously determined by checkerboard titration. The wells were coated with 100/xl of ES antigen (10 /xg ml -~) diluted in 0.1 M carbonate buffer pH 9.6 and incubated overnight at 4°C. After washes with phosphate buffer saline (PBS)-0.1% Tween 20, 100 /xl of goat sera diluted at 1:800 in PBS-10% Foetal calf serum (FCS) were added in duplicate and incubated for 30 min at room temperature. After washes, 100 /xl of horseradish peroxidase conjugated rabbit anti-goat IgG (Heavy and light chains (H &L)) (Sigma Immunologicals) was incorporated, at the dilution of 1:1000. The incubation time for the conjugate was 30 mim. After washing the conjugate, 100 /xl of the chromogen ortho-phenylenediamidine (OPD: Sigma Immunologicals) was added and incubated for 30 min. The reaction was stopped by adding 50/xl of 8 M sulphuric acid. The optical density (OD) was read at 492 nm in a SLTTM-Easy Reader microplate spectrophotometer. The results were expressed as percentage of antibody, using the following calculation (Poitou et al., 1992): [(sample mean OD) - (negative pool mean OD)/(positive pool mean OD) - (negative pool mean OD)] × 100. Serum pools from six naturally infected goats and six uninfected ones were used, respectively, as positive and negative controls. These standards were repeated in each plate. Results were tested for statistical correlation using the ANOVA test, and P values of 0.05 or lower were considered statistically significant.
3. Results and discussion
The mean values of the antibody levels obtained by ELISA during the experimental infection in the three groups are shown in Fig. 1. Considering these results we established the positive limit of the test in an antibody percentage of 15%: this cut-off level was based on the 99% percentile of negative control sera and preinfection values of all infected goats. The preinoculation values and the values of the control group throughout the experiment were lower than 0.9%. In the infected groups the antibody levels began to rise 15 days PI, and 30 days PI all goats (in both groups) showed values higher than 15%. From Day 30 until Day 360 PI, antibody levels in all infected goats were significantly higher ( P < 0.01) than in the controls. In both groups the evolution of the antibody levels was similar: there was a steady increase reaching the highest levels 90 days PI, after which the antibody levels began to decline; the decrease was especially remarkable between Day 90 and Day 145 PI, then it was slower until the end of the experiment (360 days PI), when the values were still significantly higher than in the control group.
A. Martlnez et a L / Veterinary Parasitology 62 (1996) 247-252
250 350 300
250.
~ 180. Q ~
T
~ 100. IZ 80.
rttt "80
....
I .... 30
I .... 60
I .... 90
t I .... 120
I .... 150
I .... 180 DAYS
I I .... 210
I .... 240
f I .... 270
I .... 300
I .... 330
I .... 360
390
PI
Fig. I. Means 4- standard errors of the means of antibody percentage to ES products of F. hepatica during the
course of the experimental infections. Goats infected with 200 metacercariae(B), goats infected with 100 metacercariae(0) and uninfected controls (A). Horizontalline represents the limit for positiveresults (15%).
Although in Group 1 the ELISA values were higher than in Group 2, there were no significant differences ( P > 0.05) between them and a positive correlation was observed between the curves of both groups. However, there was a significant difference ( P < 0.05) between Group 1 and 2 in the mean number of flukes recovered at necropsy: the mean in Group 1 was 25.2 and in Group 2, 10.4. F. hepatica eggs appeared in the faeces of infected animals between 60 and 90 days PI, and were recovered until the end of the experiment. There was no difference between infected groups in relation to chronology or amount of eggs eliminated. Eggs were not seen in the uninfected group. The ELISA test with ES products as antigens has been proved to be a sensitive, specific and an early method of detection of F. hepatica infection in cattle and sheep (Santiago and Hillyer, 1988; Sinclair and Wassail, 1988). We have found that the application of the ELISA with ES antigens allows the early detection of specific antibodies in goats fasciolosis and that could be an interesting diagnostic method in this animal. ES-specific antibodies were detected in significant levels between 15 and 45 days PI in all infected goats (in both groups of infection). Serological evidence of the infection was therefore much earlier than faecal examinations: eggs did not appear in the faeces until 75-95 days PI. This early detection of antibodies in goats by ELISA coincide with the early diagnosis reported by Levieux and Levieux (1994) using an HA test; it is also similar in sheep using ELISA (Santiago and Hillyer, 1988) or DOT-ELISA (Zimmerman et al., 1985) and in cattle with ELISA (Santiago and Hillyer, 1988).
A. MartEnez et al. / Veterinary Parasitology 62 (1996) 247-252
251
The dynamics of antibody production throughout the infection in both groups of goats (infected with 100 and 200 metacercafiae), as determined by the ELISA, was similar to that described by Levieux and Levieux (1994) using an HA test in a single infection with 300 metacercariae, but over a shorter period: 15 weeks (105 days). In these experimental infections of goats the maximum antibody level was reached between 60 and 105 days PI, after which there was a slow decrease that Levieux and Levieux followed for 15 weeks PI and we extended until 365 days PI (52 weeks PI). We have therefore observed the persistence of significant levels of antibodies as long as 1 year after a single primary infection. This data must be compared and contrasted with those of natural infections, because the long persistence of "positive" antibody levels could be an interesting factor to consider in seroepidemiological surveys, which may affect the best time of the year to check the animals (considering the epidemiological chronology of the infection). Using ELISA we did not observe significant differences in antibody levels of the groups infected with different doses of metacercariae (100 and 200 metacercariae). This lack of influence of the primary infective dose in the antibody response has been reported in sheep with 250 and 500 metacercariae (Zimmerman et al., 1982) but it may be related to the sizes of the infective doses tested: Wyckoff and Bradley (1986) reported significant differences in cattle infected with 3000 and 30 metacercariae, but not with 3000 and 300 or with 300 and 30. Our results highlight for the first time the possibilities and advantages of the ELISA (with ES antigen) for the diagnosis of caprine fasciolosis. Here we present results limited to primary experimental infections, but we are carrying out studies that include the use of this ELISA test in field studies, the potential cross-reactivity with other nematodes, the determination of IgM antibodies and the dynamics of production of IgG and IgM in high and low dose primary infections, reinfections and treatments.
Acknowledgements We thank Dr. C. Boulard for her technical and scientific assistance and for providing metacercariae. This research was supported by a grant from the EEC for Research and Development Program in the field of Science and Technology Development (0200F) and the project AGF92-0985 of the Comisitn Interministerial de Ciencia y Tecnologla (CICYT).
References Khalaayoune, K. and El-Haft, M., 1991. Variations saisonnieres de l'infection par Fasciola hepatica chez le chevre dans la region du Haouz (Maroc). Ann. Rech. Vet., 22: 219-226. Leathers, C.W., Foreyt, W.J., Fetcher, A. and Foreyt, K.M., 1982. Clinical fasciolosis in domestic goats in Montana. J. Am. Vet. Med. Assoc., 180: 1451-1454. Levieux, D. and Levieux, A., 1994. Early immunodiagnosis of caprine fasciolosis using the specific f2 antigen in a passive hemagglutination test. Vet. Parasitol., 53: 59-66.
252
A. Marffnez et al. / Veterinary Parasitology 62 (1996) 247-252
Lowry, O.H., Rosenbrough, N.J., Fair, A.L. and Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Poitou, I., Baeza, E. and Boulard, C., 1992. Humoral and cellular immune responses in rats during a primary infection with Fasciola hepatica. Vet. Parasitol., 45: 59-71. Reddington, J.J., Leid, R.W. and Wescott, R.B., 1986. The susceptibility of the goat to Fasciola hepatica infections. Vet. Parasitol., 19: 145-150. Santiago, N. and Hillyer, G.V., 1988. Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasclola hepatica. J. Parasitol., 74: 810-818. Schenone, H. and Rojas, A., 1988. Epidemiologla de la fasciolosis animal en Chile. Tendencia de las tasas de infecci6n, por regiones, en cinco especies de mamfferos de consumo beneficiados en mataderos del pals (1976-1977). Boletln Chileno de Parasitologia, 43: 68-70. Sharma, R.L., Dhar, D.N. and Raina, O.K., 1989. Studies on the prevalence and laboratory transmission of fasciolosis in animals in the Kashmir valley. Br. Vet. J., 145: 57-61. Sinclair, J.J. and Wassail, D.A., 1988. Serodiagnosis of Fasciola hepatica infections in cattle. Vet. Parasitol., 27: 283-290. Thienpont, D., Rochette, F. and Vanparijs, O.F.J., 1986. Diagnosis of Helminthiasis by Coprological Examinations. Janssen Research Foundations, Beerse, Belgium. Topadak, M. and Giill, Y., 1988. Liver fluke infections in goats slaughtered at Van abattoir. Veterinary Fakultesi Dergisi, Ankara Univeresitesi, 35: 412-417. Wang, P.Y., Jin, J.S., Cai, X.P., Duan, Z.Q. and Yuan, W.Z., 1987. A regional trail of the ELISA for diagnosis fasciolosis in sheep and goats. Chin. J. Vet. Sci. Technol., 8: 3-7. Wyckoff II1, J.H. and Bradley, R.E., 1986. An optimized enzyme immunosorbent assay for quantitative diagnosis of bovine fasciolosis. J. Parasitol., 72: 439-444. Zimmerman, G.L., Jen, L.W., Cerro, J.E., Farnsworth, K.L. and Wescott, R.B., 1982. Diagnosis of Fasciola hepatica in sheep by an enzyme linked immunosorbent assay. Am. J. Vet. Res., 43: 2097-2100. Zimmerman, G.L., Nelson, M.J. and Clark, C.R.B., 1985. Diagnosis of ovine fasciolosis by a dot-enzyme immunosorbent assay: a rapid microdiagnostic technique. Am. J. Vet. Res., 46: 1513-1515.
Veterinary Parasitology 62 (I 996) 247-252
Detection of antibodies to Fasciola hepatica excretory-secretory antigens in experimentally infected goats by enzyme immunosorbent assay A. Martfnez *, M.S. Marffnez-Cruz, F.J. Marffnez, P.N. Gutierrez, S. Hemg.ndez Cdtedra de Parasitologlay Enfermedades Parasitarias, Departamentode Sanidad Animal, Facultad de Veterinaria, Universidad de Ctrdoba, 14071 Ctrdoba, EspaYza Received 12 April 1995; accepted 10 July 1995
Abstract An EL1SA with excretory-secretory (ES) antigens has been evaluated as a technique for the early detection of specific antibodies in Fasciola hepatica infections in goats. Goats were experimentally infected with 100 or 200 metacercariae of bovine origin and serum samples were taken periodically over 365 days. The ELISA test was performed with ES antigens (10/xg ml-t), a single dilution of sera (1:800) and anti-goat lgG conjugate (1:1000). ES specific antigens were detected in all infected goats between 15 and 30 days postinfection (PI) and maximum antibody levels were reached at 90 days PI. Positive antibody levels (significantly different from those of controls) were still found at 365 days PI. No significant differences were observed between goats infected with 100 or 200 metacereariae. In all infected goats, eggs appeared in faeces between 60 and 90 days PI. ELISA with ES antigens could be a feasible method for the early diagnosis of goat fasciolosis. Keywords: Goat; Fasciola hepatica; Diagnosis-Trematoda; ELISA
1. I n t r o d u c t i o n Fasciolosis in goats is considered to be a less frequent and less important infection than in cattle and sheep in Europe. However, it is a common infection in many areas of the world: prevalences between 72% in China (Wang et al., 1987), 14% in India
*Corresponding author. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 8 7 6 - 4
248
A. Martinez et al. / Veterinary Parasitology 62 (1996) 247-252
(Sharma et al., 1989) 20% in Chile (Schenone and Rojas, 1988) 18% in Turkey (Toparlak and Giill, 1988) and 20% in Morocco (Khalaayoune and E1-Hari, 1991) have been already reported. Goats have been described as extremely sensitive to natural and experimental infections (Reddington et al., 1986). The disease usually appears in a chronic form but acute fasciolosis with high rates of mortality has been described (Leathers et ai., 1982). As treatment in the initial stages of the infection considerably reduces liver injury, it is therefore desirable to have a simple, sensitive and specific test for the early diagnosis of fasciolosis in goats. Although several immunological tests have been developed for the detection of specific antibodies to F. hepatica infections in cattle and sheep, few of them have been evaluated in goats: only Levieux and Levieux (1994) reported a haemagluttination (HA) test for the early diagnosis of caprine fasciolosis. The ELISA is the most widely used test, because of its simplicity, reliability and easy mechanization, but it has not been used in goats. The ELISA for cattle and sheep has been proved to be sensitive, specific, rapid and easy to perform (Zimmerman et al., 1982). Improved sensitivity and specificity has been obtained with the purification of somatic antigens and the use of excretory-secretory (ES) products of F. hepatica as antigen (Santiago and Hillyer, 1988). The aim of our work was to evaluate the ELISA with ES antigens as a technique for the early detection of specific antibodies to F. hepatica infections in goats, evaluating the dynamics of antibody production throughout the infection and its correlation with faecal egg output. We have used primary experimental infections with two different infective doses of metacercariae and followed the goats for 1 year.
2. Materials and methods
Eighteen "Serrana" breed males, aged 6 months were used in the study. They were left for 3 months to acclimatize to the experimental conditions. The animals were examined to exclude the possibility of any previous F. hepatica infection by coprological and serological methods. They were kept in stalls and fed with hay and a commercial pelleted ration containing 16.5% proteins. Water was available ad libitum. F. hepatica metacercariae were obtained from Lymnea truncatula snails infected with eggs collected from naturally infected cattle. The metacercariae were kept at 4°C for 6 months until used. The goats were divided in three groups: Group 1 consisted of 6 animals infected with 200 metacercariae; Group 2, of 6 animals infected with 100 metacercariae and Group 3, of 6 animals left as uninfected controls. The metacercariae were administered orally in a 5 ml saline suspension. Blood and faecal samples were taken from each animal on Days 0, 15, 30, 45, 60, 75, 90, 105, 125, 145, 195, 245, 305 and 365 pos infection (PI). Serum was separated and stored at - 8 0 ° C until used. Faecal examinations were performed by a sedimentation method (Thienpont et al., 1986)
A. Martfnez et al. / Veterinary Parasitology 62 (1996) 247-252
249
The F. hepatica ES antigen was obtained as described by Poitou et al. (1992). Briefly, adult liver flukes obtained from cattle at the slaughterhouse were washed and finally incubated in physiological saline (pH 7.2) at 37°C for 24 h. The solution was centrifuged at 4°C and 20000 rpm for 30 min to remove eggs and particulate material. The supernatant containing excretory-secretory products (ES) was collected and its protein concentration was measured as described by Lowry et al. (1951). The ELISA was performed in 96 wells microtiter plates (Maxisorp, Nunc). The optimum antigen, serum and conjugate concentrations and the incubation times were previously determined by checkerboard titration. The wells were coated with 100/xl of ES antigen (10 /xg ml -~) diluted in 0.1 M carbonate buffer pH 9.6 and incubated overnight at 4°C. After washes with phosphate buffer saline (PBS)-0.1% Tween 20, 100 /xl of goat sera diluted at 1:800 in PBS-10% Foetal calf serum (FCS) were added in duplicate and incubated for 30 min at room temperature. After washes, 100 /xl of horseradish peroxidase conjugated rabbit anti-goat IgG (Heavy and light chains (H &L)) (Sigma Immunologicals) was incorporated, at the dilution of 1:1000. The incubation time for the conjugate was 30 mim. After washing the conjugate, 100 /xl of the chromogen ortho-phenylenediamidine (OPD: Sigma Immunologicals) was added and incubated for 30 min. The reaction was stopped by adding 50/xl of 8 M sulphuric acid. The optical density (OD) was read at 492 nm in a SLTTM-Easy Reader microplate spectrophotometer. The results were expressed as percentage of antibody, using the following calculation (Poitou et al., 1992): [(sample mean OD) - (negative pool mean OD)/(positive pool mean OD) - (negative pool mean OD)] × 100. Serum pools from six naturally infected goats and six uninfected ones were used, respectively, as positive and negative controls. These standards were repeated in each plate. Results were tested for statistical correlation using the ANOVA test, and P values of 0.05 or lower were considered statistically significant.
3. Results and discussion
The mean values of the antibody levels obtained by ELISA during the experimental infection in the three groups are shown in Fig. 1. Considering these results we established the positive limit of the test in an antibody percentage of 15%: this cut-off level was based on the 99% percentile of negative control sera and preinfection values of all infected goats. The preinoculation values and the values of the control group throughout the experiment were lower than 0.9%. In the infected groups the antibody levels began to rise 15 days PI, and 30 days PI all goats (in both groups) showed values higher than 15%. From Day 30 until Day 360 PI, antibody levels in all infected goats were significantly higher ( P < 0.01) than in the controls. In both groups the evolution of the antibody levels was similar: there was a steady increase reaching the highest levels 90 days PI, after which the antibody levels began to decline; the decrease was especially remarkable between Day 90 and Day 145 PI, then it was slower until the end of the experiment (360 days PI), when the values were still significantly higher than in the control group.
A. Martlnez et a L / Veterinary Parasitology 62 (1996) 247-252
250 350 300
250.
~ 180. Q ~
T
~ 100. IZ 80.
rttt "80
....
I .... 30
I .... 60
I .... 90
t I .... 120
I .... 150
I .... 180 DAYS
I I .... 210
I .... 240
f I .... 270
I .... 300
I .... 330
I .... 360
390
PI
Fig. I. Means 4- standard errors of the means of antibody percentage to ES products of F. hepatica during the
course of the experimental infections. Goats infected with 200 metacercariae(B), goats infected with 100 metacercariae(0) and uninfected controls (A). Horizontalline represents the limit for positiveresults (15%).
Although in Group 1 the ELISA values were higher than in Group 2, there were no significant differences ( P > 0.05) between them and a positive correlation was observed between the curves of both groups. However, there was a significant difference ( P < 0.05) between Group 1 and 2 in the mean number of flukes recovered at necropsy: the mean in Group 1 was 25.2 and in Group 2, 10.4. F. hepatica eggs appeared in the faeces of infected animals between 60 and 90 days PI, and were recovered until the end of the experiment. There was no difference between infected groups in relation to chronology or amount of eggs eliminated. Eggs were not seen in the uninfected group. The ELISA test with ES products as antigens has been proved to be a sensitive, specific and an early method of detection of F. hepatica infection in cattle and sheep (Santiago and Hillyer, 1988; Sinclair and Wassail, 1988). We have found that the application of the ELISA with ES antigens allows the early detection of specific antibodies in goats fasciolosis and that could be an interesting diagnostic method in this animal. ES-specific antibodies were detected in significant levels between 15 and 45 days PI in all infected goats (in both groups of infection). Serological evidence of the infection was therefore much earlier than faecal examinations: eggs did not appear in the faeces until 75-95 days PI. This early detection of antibodies in goats by ELISA coincide with the early diagnosis reported by Levieux and Levieux (1994) using an HA test; it is also similar in sheep using ELISA (Santiago and Hillyer, 1988) or DOT-ELISA (Zimmerman et al., 1985) and in cattle with ELISA (Santiago and Hillyer, 1988).
A. MartEnez et al. / Veterinary Parasitology 62 (1996) 247-252
251
The dynamics of antibody production throughout the infection in both groups of goats (infected with 100 and 200 metacercafiae), as determined by the ELISA, was similar to that described by Levieux and Levieux (1994) using an HA test in a single infection with 300 metacercariae, but over a shorter period: 15 weeks (105 days). In these experimental infections of goats the maximum antibody level was reached between 60 and 105 days PI, after which there was a slow decrease that Levieux and Levieux followed for 15 weeks PI and we extended until 365 days PI (52 weeks PI). We have therefore observed the persistence of significant levels of antibodies as long as 1 year after a single primary infection. This data must be compared and contrasted with those of natural infections, because the long persistence of "positive" antibody levels could be an interesting factor to consider in seroepidemiological surveys, which may affect the best time of the year to check the animals (considering the epidemiological chronology of the infection). Using ELISA we did not observe significant differences in antibody levels of the groups infected with different doses of metacercariae (100 and 200 metacercariae). This lack of influence of the primary infective dose in the antibody response has been reported in sheep with 250 and 500 metacercariae (Zimmerman et al., 1982) but it may be related to the sizes of the infective doses tested: Wyckoff and Bradley (1986) reported significant differences in cattle infected with 3000 and 30 metacercariae, but not with 3000 and 300 or with 300 and 30. Our results highlight for the first time the possibilities and advantages of the ELISA (with ES antigen) for the diagnosis of caprine fasciolosis. Here we present results limited to primary experimental infections, but we are carrying out studies that include the use of this ELISA test in field studies, the potential cross-reactivity with other nematodes, the determination of IgM antibodies and the dynamics of production of IgG and IgM in high and low dose primary infections, reinfections and treatments.
Acknowledgements We thank Dr. C. Boulard for her technical and scientific assistance and for providing metacercariae. This research was supported by a grant from the EEC for Research and Development Program in the field of Science and Technology Development (0200F) and the project AGF92-0985 of the Comisitn Interministerial de Ciencia y Tecnologla (CICYT).
References Khalaayoune, K. and El-Haft, M., 1991. Variations saisonnieres de l'infection par Fasciola hepatica chez le chevre dans la region du Haouz (Maroc). Ann. Rech. Vet., 22: 219-226. Leathers, C.W., Foreyt, W.J., Fetcher, A. and Foreyt, K.M., 1982. Clinical fasciolosis in domestic goats in Montana. J. Am. Vet. Med. Assoc., 180: 1451-1454. Levieux, D. and Levieux, A., 1994. Early immunodiagnosis of caprine fasciolosis using the specific f2 antigen in a passive hemagglutination test. Vet. Parasitol., 53: 59-66.
252
A. Marffnez et al. / Veterinary Parasitology 62 (1996) 247-252
Lowry, O.H., Rosenbrough, N.J., Fair, A.L. and Randall, R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Poitou, I., Baeza, E. and Boulard, C., 1992. Humoral and cellular immune responses in rats during a primary infection with Fasciola hepatica. Vet. Parasitol., 45: 59-71. Reddington, J.J., Leid, R.W. and Wescott, R.B., 1986. The susceptibility of the goat to Fasciola hepatica infections. Vet. Parasitol., 19: 145-150. Santiago, N. and Hillyer, G.V., 1988. Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasclola hepatica. J. Parasitol., 74: 810-818. Schenone, H. and Rojas, A., 1988. Epidemiologla de la fasciolosis animal en Chile. Tendencia de las tasas de infecci6n, por regiones, en cinco especies de mamfferos de consumo beneficiados en mataderos del pals (1976-1977). Boletln Chileno de Parasitologia, 43: 68-70. Sharma, R.L., Dhar, D.N. and Raina, O.K., 1989. Studies on the prevalence and laboratory transmission of fasciolosis in animals in the Kashmir valley. Br. Vet. J., 145: 57-61. Sinclair, J.J. and Wassail, D.A., 1988. Serodiagnosis of Fasciola hepatica infections in cattle. Vet. Parasitol., 27: 283-290. Thienpont, D., Rochette, F. and Vanparijs, O.F.J., 1986. Diagnosis of Helminthiasis by Coprological Examinations. Janssen Research Foundations, Beerse, Belgium. Topadak, M. and Giill, Y., 1988. Liver fluke infections in goats slaughtered at Van abattoir. Veterinary Fakultesi Dergisi, Ankara Univeresitesi, 35: 412-417. Wang, P.Y., Jin, J.S., Cai, X.P., Duan, Z.Q. and Yuan, W.Z., 1987. A regional trail of the ELISA for diagnosis fasciolosis in sheep and goats. Chin. J. Vet. Sci. Technol., 8: 3-7. Wyckoff II1, J.H. and Bradley, R.E., 1986. An optimized enzyme immunosorbent assay for quantitative diagnosis of bovine fasciolosis. J. Parasitol., 72: 439-444. Zimmerman, G.L., Jen, L.W., Cerro, J.E., Farnsworth, K.L. and Wescott, R.B., 1982. Diagnosis of Fasciola hepatica in sheep by an enzyme linked immunosorbent assay. Am. J. Vet. Res., 43: 2097-2100. Zimmerman, G.L., Nelson, M.J. and Clark, C.R.B., 1985. Diagnosis of ovine fasciolosis by a dot-enzyme immunosorbent assay: a rapid microdiagnostic technique. Am. J. Vet. Res., 46: 1513-1515.