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Antibody and circulating antigen profiles before and after chemotherapy in goats infected with Fasciola gigantica
veterinary parasitology ELSEVIER
Veterinary Parasitology66 (1996) 171 179 -
Antibody and circulating antigen profiles before and after chemotherapy in goats infected with Fasciola gigantica Judith V. Mbuh, Benjamin O. Fagbemi * Department of Veterinary Microbiology and Parasitology, University of lbadan, lbadan, Nigeria Received 16 January 1996; accepted 5 April 1996
Abstract The profiles of antibody response and circulating antigen levels in goats infected with Fasciola gigantica were studied by enzyme-linked immunoelectrotransfer blot (EITB) and enzyme-linked immunosorbent assay (ELISA). In the antibody assay, sera from goats experimentally infected with F. gigantica were reacted with whole worm antigen of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in antibody level 2 weeks after infection. After chemotherapy, there was a gradual decrease in antibody within 3 weeks followed by a rapid decline by the 4th week after treamaent. By EITB, the infected goat sera recognized three polypeptides in the range of 42-80 kDa as early as 2 weeks after infection. Recognition of the complete components of F. gigantica antigen repertoire occurred as early as the 4th week after infection. By the 8th week after chemotherapy, distinct polypeptide band recognition was no longer possible. Comparative immunoblottingwith goat anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 14 kDa, 17 kDa, 21 kDa, 28 kDa and 30 kDa proteins are specific to F. gigantica. In the antigen assay, circulating antigen was detectable by the direct ELISA method one week after infection and negative absorbance values were observed 4 weeks after chemotherapy.
Keywords: Fasciola gigantica; Goat-Trematoda; Immunoblot;Circulatingantigen
1. Introduction
Fasciola gigantica is an important parasite of all domestic ruminants but while the infection in cattle and sheep is well known, fascioliasis in goats has not been the subject
* Correspondingauthor. 0304-4017/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0304-4017(96)01019-9
172
J. V. Mbuh. B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
of much study. Furthermore, it is known that the course of fascioliasis in the host varies according to the ruminant species. For example, fascioliasis is often chronic and subclinical in cattle, but acute and accompanied by high mortality in sheep (Soulsby, 1982). Furthermore, the immunological consequences of fascioliasis vary among different ruminant species. While cattle acquire some degree of resistance following a primary infection (Ross, 1966; Boray, 1969; Doyle, 1971; Doyle, 1973), sheep seem to remain highly susceptible to fascioliasis after previous infections (Sinclair, 1975; Knight, 1980; Sandeman and Howell, 1981). The situation in goats is not known. The differences in the severity of fascioliasis and immunological responses among different ruminant species was considered to be partly attributable to variations in time-course responsiveness of these hosts to different antigenic determinants of the parasite. Profound antigenic alterations are associated with the development of the worm from a juvenile fluke to egg-laying adult (Bennet and Threadgold, 1975; Rajasekariah and Howell, 1978; Hanna, 1980a; Hanna, 1980b; Sandeman and Howell, 1981; Bennet et al., 1982). This implies a corresponding variation in the immunological reaction of the host in the course of an infection. The time-course analysis of antibody response in sheep infected with F. gigantica has been studied recently (Guobadia and Fagbemi, 1995). In this paper, we have investigated the sequence of antibody response and the profile of circulating antigen in goats infected with F. gigantica by immunoblotting and ELISA before and after chemotherapy. In addition, we have identified the antigens which have potential usefulness in immunodiagnosis of the fluke infection in goats.
2. Materials and methods
2.1. Preparation of antigen Live, adult F. gigantica, Paramphistomum microbothrium and Dicrocoelium hospes were collected from goats slaughtered in a local abattoir. They were washed four times with 0.01 M phosphate buffered saline (PBS) pH 7.2 to remove all traces of bile and blood. Whole worm antigen of F. gigantica was prepared by homogenizing the worms (1 worm ml- ~ PBS) in PBS which was supplemented wih 0.25 mg ml- ~ leupeptin in a Ten Broeck tissue grinder in an ice bath. The leupeptin served as a protease inhibitor. The mixture was centrifuged at 10000 g for 20 min. The supernatants were stored at - 2 0 ° C until used. Whole worm antigen extracts of P. microbothrium (PmWWE) and D. hospes (DhWWE) were prepared in the same way. The protein concentrations were determined by the procedure described by Bradford (1976).
2.2. Experimental animals Six, 6-month-old goats which were rared under Fasciola free conditions and ascertained free of infection by coprological examination were used. Four of the goats were infected orally with 350 metacercariae of F. gigantica each. Two goats served as uninfected controls. Blood samples were collected at weekly intervals from the jugular vein of infected
J.V. Mbuh, B.O. Fagbemi/ Veterinary Parasitology 66 (1996) 171-179
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goats for 12 weeks. On the 12th week the goats were treated with 33 mg kg -~ oxyclozanide (Vermofas, Bimeda, Dublin) to terminate the infection. Thereafter, serum samples were collected for another 12 weeks. Serum samples from non-infected goats served as controls. A second group of goats were immunized with either PmWWE or DhWWE (10 mg antigen) in Freund's complete adjuvant. Booster doses containing 5 mg antigen in Freund's incomplete adjuvant were given three times at weekly intervals. Serum samples were collected one week after the last immunization. All samples were stored at - 20°C. Faecal samples of infected animals were examined at weekly intervals for the presence of F. gigantica eggs by the sedimentation method of Thienpont et al. (1979). Three rabbits were each infected orally with 15 metacercariae of F. gigantica. Blood was collected after 14 weeks from the ear vein.
2.3. Enzyme-linked immunoelectrotransfer blot (EITB) The method used was that described by Tsang et al. (1983) and Santiago and Hillyer (1986). Antigens were prepared at 4 ml- 1 in a sample buffer of 0.5 M Tris-HC1, 10% glycerol, 10% sodium dodecyl sulphate, 5% B-mercaptoethanol and 0.1% bromophenol blue. Whole worm extracts of F. gigantica antigens were electrophoresed in 12.5% polyacrylamide gel at 150 volts for 1½ h in a protein 11 minigel apparatus (BIORAD). Transfer of proteins from gel to nitrocellulose sheets was performed at 30 volts overnight at 4°C. The nitrocellulose sheet was cut into strips and incubated with the test sera which were diluted 1:200 with 0.05% PBS-Tween 20. The test sera consisted of those from goats with experimental F. gigantica infection, anti-PmWWE and antiDhWWE. This was followed by incubation in a 1:500 dilution of a horseradish peroxidase conjugated rabbit anti-goat IgG in 0.3% PBS-Tween 20. The strips were developed by the addition of 3, 3'-diaminobenzidine tetrahydrochloride (Sigma).
2.4. Enzyme linked immunosorbent assay (ELISA) ELISA was done as described by Santiago et al. (1986). Optimum working dilution of antigen as determined by checkerboard titration was 5 mg ml -I. The antigen was diluted in carbonate buffer pH 9.3 and incubated overnight at 4°C. Test sera were used at 1:200 dilution in 1% skimmed milk in 0.3% PBS-Tween 20. This was followed by incubation for 2 h at 37~C. Horseradish peroxidase conjugated anti-goat IgG (Kirkegaard and Perry Laboratories, Gaithensburg, MD) at a dilution of 1:1000 in 0.3% P B S / T w e e n 20 was then incubated for 1 h. O-phenylene diamine was used as substrate and incubated for 30 min at room temperature in the dark. A solution of 0.25 M H2SO 4 was added (50 I~1 per well) to terminate the reaction. Absorbance values were read spectrophotometrically at 492 nm.
2.5. Conjugation of rabbit anti-Fasciola serum to horseradish peroxidase Sera obtained from rabbits infected with F. gigantica was conjugated to horseradish peroxidase by the glutaraldehyde method (Lunde et al., 1987).
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Briefly, 10 ml of rabbit anti-F, gigantica antiserum was added to 2.5 g ammonium sulphate for 20 h at room temperature. The mixture was centrifuged at 10000 g for 1 h at 4°C. The immunoglobulin precipitate was washed three times with 25 ml of 1.75 M ammonium sulphate and centrifuged three times. One ml of distilled water was added to the precipitate and it was dialysed at 4°C for 24 h against 0.050 M sodium acetate/0.021 M acetic acid pH 5.0, and finally against 0.1 M sodium chloride/sodium azide for 24 h. A total of 125 txl of glutaraldehyde was added to 10 ml of PBS. Horseradish peroxidase (0.01 g) was added to 200 ill of the glutaraldehyde mixture and left at room temperature. One ml of 0.1 M carbonate buffer (pH 7.2) was added to the glutaraldehyde mixture. This was dialysed against carbonate buffer (pH 9.2), changing twice in 4 h. Five mg of the F. gigantica immunoglobulin was added to the glutaraldehyde mixture containing peroxidase; 0.1 ml of 0.2 M lysine was then added, together with an equal volume of glycerol. This preparation was stored at 20°C until used.
2.6. Detection of circulating F. gigantica antigen using the direct ELISA method A total of 100 I~1 of goat sera from experimentally infected goats were plated in each well of ELISA plates overnight at 4°C. The plates were washed five times at 15 min intervals with 0.05% PBS-Tween 20, after which the unreacted binding sites were blocked by incubating the plates with 1% skimmed milk in 0.3% PBS Tween 20 for 1 h at 37°C. Incubation with peroxidase labelled rabbit anti-Fasciola IgG (1:1000 dilution) was for lh. After five washes of 15 min each, 100 i~1 of the substrate, O-phenylene-diamine (OPD) was added and incubated for 30 min in the dark for colour development. The reaction was stopped by adding 50 I~1 of 1 M H2SO 4 to each well. The absorbance was read spectrophotometrically at 492 nm. The cut-off point to define a positive or negative serum sample was established as the mean absorbance of the normal sera + 2 SD.
POST INFECTION 2 4 681012
POST TREATMENT 2 4 6 8 1 0 1 2 (:
90-7g-5g42352g14Fig. 1. Immunoblot of F. gigantica somatic antigen with sera of infected goats before and after chemotherapy. The sera of six goats were pooled each week for the test.
J.V. Mbuh, B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
F
O
175
p
95---62 55 3028
-
21,----14-----
Fig. 2. Comparative immunobiot of F. gigantica somatic antigen with goat anti-Dicrocoelium (D), anti-Fasciola (F) and anti-Paramphistomum (P) sera. The anti-F sera were pooled from goats with 10 week old F. gigantica infection.
1.0CHEMOTHERAPY
0.8
0.6
0.4
o_~__ < 0.2-
0.0 3
5'
7'
9"
~ 11
' 13
~ 15
I ~7
~ 19
~ 21
2 ~3
' 25
Time (weeks)
Fig. 3. ELISA absorbance values for sera of F. gigantica infected goats before and after chemotherapy with oxyclozanide. Horizontal broken line indicates mean absorbance of normal sera + 2SD = 0.25.
J.V. Mbuh, B.O. Fagbemi/ VeterinaryParasitology66 (1996) 171-179
176 3. Results
Fasciola eggs were detected in the faeces of the infected goats 10 weeks after infection and they were not detected 1 week after chemotherapy. In EITB, three polypeptides of molecular weight 80 kDa, 59 kDa and 44 kDa were recognized as early as 2 weeks after infection (Fig. 1). By the 4th week, more polypeptide bands ranging in molecular weight from 17 kDa to 90 kDa were recognized. The 29 kDa, 35 kDa and 59 kDa polypeptides were more intensely recognized as from the 6th week. The recognition of these protein bands persisted until the 12th week after infection. By 4 weeks after chemotherapy, recognition of most of the protein bands had ceased although faint bands were still noticed at Week 6. By 12 weeks the bands that could be seen were diffused and non distinctive and no definite banding pattern was observed. A comparative immunoblotting study of F. gigantica whole worm extract with anti-P, microbothrium, anti-D, hospes and anti-F, gigantica revealed cross-reactivities. Anti-D. hospes serum recognized bands between 55-90 kDa, while anti-P, microbothrium serum recognized protein bands of molecular weight between 44-90 kDa and anti-F, gigantica serum recognized bands ranging between 14-92 kDa. The bands that appeared to be specific to F. gigantica were 30 kDa, 28 kDa, 21 kDa, 17 kDa and 14 kDa (Fig. 2).
CHE M O T H E R A P Y
(I.5
E
]
3
5
7
9
11
13
15
17
19
21
23
25
']'ir,',e ( w e e k s )
Fig. 4, ELISAresults of circulating F. gigantica antigens of experimentally infected goals before and after chemotherapy. Horizontal broken line indicates mean absorbance of normal sera + 2SD = 0.15. Each point represents the mean of three replicates.
J.V. Mbuh, B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
177
ELISA showed a steady antibody increase as from 2 weeks post infection until Week 12 when an absorbance of 0.8 was obtained (Fig. 3). After chemotherapy, antibody levels started to fall as from Week 13. There was a sharp decline between Weeks 15 and 16, followed by a steady fall in absorbance until Week 24. In the antibody-detection assay, circulating Fasciola antigens were detected by Week l after infection. The peak absorbance of these antigens was detected from Week 6 after infection to Week 12 when chemotherapy was done. At 4 Weeks after chemotherapy, no circulating antigen was detected till the end of the experiment (Fig. 4).
4. Discussion
In this study, the antibody and circulating antigen profiles were studied by EITB and ELISA after infecion of goats with F. gigantica. The detectable antibody response observed 2 weeks after infection in ELISA is consistent with the trend observed in sheep infected with the flukes (Guobadia and Fagbemi, 1995). Moreover, the rapid decline in antibody level after chemotherapy falls in line with the observation in sheep. In this way, the patterns of antibody response to F. gigantica infection in goats and sheep are similar to that which was reported in sheep infected with F. hepatica (Zimmerman et al., 1982; Santiago and Hillyer, 1988). However, in immunoblotting studies, while there was a gradual increase in the number of polypeptide bands of F. gigantica recognized by infected sheep sera in the weeks after infection (Guobadia and Fagbemi, 1995), the number of antigen bands recognized by infected goat sera appeared to reach a maximum 4 weeks after infection. There was a rapid immune recognition of all the components of the complete antigen repertoire of F. gigantica by infected goat sera within 4 weeks after infection. Thereafter, differences in recognition was only a matter of intensity. This contrasts sharply with what was observed in sheep in which the 87 kDa antigen was recognized as late as 12 weeks after infection (Guobadia and Fagbemi, 1995). The rapid immune recognition of F. gigantica antigens by goats may have much significance in determining the course of the infection and in defining the degree of immunity in this species. Furthermore, the early recognition of these antigens increases their usefulness for immunodiagnosis in the early stage of infection. Comparative immunoblotting with anti-P, microbothrium and anti-D, hospes sera revealed that at least four antigens including the 17 kDa and 21 kDa seem to be specific to F. gigantica. Polypeptide antigens of the same 17 and 21 kDa molecular weights have been reported to be specific to F. gigantica in sheep (Guobadia and Fagbemi, 1995). These antigens are therefore potentially good immunodiagnostic antigens. However, it should be realized that immunization of goats with whole worm antigen of P. microbothrium and D. hospes may not produce the real representation of the normal immunological pattern that may be obtained after the infection of the animals by these trematodes. Circulating antigens normally show active and present infections. The disappearance of antigens 4 weeks after treatment is a good indication that the test is highly sensitive and will be a good immunodiagnostic assay. While in goats the antigens disappeared 4
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weeks post treatment, in sheep these disappeared 3 weeks after treatment (Guobadia and Fagbemi, 1996). The immunological recognition of the polypeptide antigen of F. gigantica, especially the specific 17 KDa and 21 KDa antigens, by infected goat sera, occurs as early as 4 weeks after infection while coproiogical diagnosis is possible only after 10 weeks of infection. Since an assay based on the detection of these specific antigens will provide an attractive alternative to the conventional diagnosis of F. gigantica in goats and sheep, work is in progress in our laboratory to purify and use the 17 KDa and 21 KDa antigens for this purpose.
Acknowledgements This study was supported by Grant No B / I 981-1 from the International Foundation for Sciences, Sweden.
References Bennet, C.E., Joshua, G.W.P. and Hughes, D.L., 1982. Demonstration of juvenile-specific antigens of Fasciola hepatica. J. Parasitol., 68: 791-795. Bermet, C.E. and Threadgold, L.T., 1975. F.hepatica: development of tegument during migration in mouse. Exp. Parasitol., 38: 38-55. Boray, J.C., 1969. Experimental fascioliasis in Australia. Adv. Parasitol., 7: 95-210. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Ann. Biochem., 72: 245-255. Doyle, J.J., 1971. Acquired immunity to experimental infection with F. hepatica in cattle. Res. Vet. Sci., 12: 529. Doyle, J.J., 1973. The relationship between the duration of a primary infection and the subsequent development of an acquired resistance to experimental infections with F. hepatica in calves. Res. Vet. Sci., 14: 97-103. Guobadia, E.E. and Fagbemi, B.O., 1995. Time-course analysis of antibody response in EITB and ELISA bebore and after chemotherapy in sheep infected with F. gigantica. Vet. Parasitol., 58: 247-253. Guobadia, E.E. and Fagbemi, B.O., 1996. Detection of circulating F. gigantica antigens in experimental and natural infections of sheep with fascioliasis. Vet. Parasitol., 65: 29-39. Hanna, R.E.B., 1980a. Fasciola gigantica: glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. Exp. Parasitol., 50:103-114. Hanna, R.E.B., 1980b. Fasciola hepatica: an immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep. Exp. Parasitol., 50: 155-170. Knight, R.A., 1980. Response of lambs to challenge infections after repeated inoculations with F. gigantica cyst. Proc. Helm. Soc. Wash., 47: 186-191. Lunde, M,, Paranjape, R., Lawley, T. and Ohessen, E.A., 1987. Filaria antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis. Am. Soc. Trop. Med. Hyg., 34: 366-371. Rajasekafiah, G.R. and Howell, M.J., 1978. F. hepatica: role of the developmental stages in the rat's resistance to challenge. Exp. Parasitol., 44: 233-238. Ross, J.G., 1966. Studies of immunity to F. hepatica. Mutually acquired immunity in rabbits. Br. Vet. J., 122: 209-211. Sandeman, R.M. and Howell, M.J., 1981. Response of sheep to challenge infection with F. hepatica. Res. Vet. Sci., 30: 294-297.
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Santiago, N. and HiUyer, G.V., 1986. Isolation of potential serodiagnostic F. hepatica antigens by electroelution from polyacrylamide gels. Am. J. Trop. Med. Hyg., 35 (6): 1210-1217. Santiago, N., Hillyer, G.V., Garcia-Rosa, M. and Morales, M.H., 1986. Identification of functional Fasciola hepatica antigens in experimental infections in rabbits. Int. J. Parasitol., 14: 197-206. Santiago, N. and Hillyer, G.V., 1988. Antibody profile by EITB and ELISA of cattle and sheep infected with F. gigantica. J. Parasitol., 74: 810-818. Sinclair, K.B., 1975. The resistance of sheep to Fasciola hepatica. Studies on the pathophysiology of challenge infections. Res. Vet. Sci., 19: 269-303. Sonisby, E.J,, 1982. Helminths, Arthropods, and Protozoa of Domesticated Animals. Bailliere Tindall, London, 809 pp. Thienpont, D., Rochete, F. and Vanprijs, O.F., 1979. Diagnosing hehninthiasis through coprological examination. Janssen Research Foundation, Beerse, 180 pp. Tsang, V.C., Peralta, J.M. and Simmons, A.R., 1983. Enzyme-linked immunoelectrotransfer blot (EITB) technique for studying the specificities and antibodies separated by gel electrophoresis. Methods Enzymol., 92: 377-391. Zimmerman, G.L., Jen, L.W., Cerro, J.E., Fransworth, K.L. and Wescott, R.B., 1982. Diagnosis of Fasciola hepatica in sheep by enzyme-linked immunosorbent assay. Am. J. Vet. Res., 43: 2097-2100.
Veterinary Parasitology66 (1996) 171 179 -
Antibody and circulating antigen profiles before and after chemotherapy in goats infected with Fasciola gigantica Judith V. Mbuh, Benjamin O. Fagbemi * Department of Veterinary Microbiology and Parasitology, University of lbadan, lbadan, Nigeria Received 16 January 1996; accepted 5 April 1996
Abstract The profiles of antibody response and circulating antigen levels in goats infected with Fasciola gigantica were studied by enzyme-linked immunoelectrotransfer blot (EITB) and enzyme-linked immunosorbent assay (ELISA). In the antibody assay, sera from goats experimentally infected with F. gigantica were reacted with whole worm antigen of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in antibody level 2 weeks after infection. After chemotherapy, there was a gradual decrease in antibody within 3 weeks followed by a rapid decline by the 4th week after treamaent. By EITB, the infected goat sera recognized three polypeptides in the range of 42-80 kDa as early as 2 weeks after infection. Recognition of the complete components of F. gigantica antigen repertoire occurred as early as the 4th week after infection. By the 8th week after chemotherapy, distinct polypeptide band recognition was no longer possible. Comparative immunoblottingwith goat anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 14 kDa, 17 kDa, 21 kDa, 28 kDa and 30 kDa proteins are specific to F. gigantica. In the antigen assay, circulating antigen was detectable by the direct ELISA method one week after infection and negative absorbance values were observed 4 weeks after chemotherapy.
Keywords: Fasciola gigantica; Goat-Trematoda; Immunoblot;Circulatingantigen
1. Introduction
Fasciola gigantica is an important parasite of all domestic ruminants but while the infection in cattle and sheep is well known, fascioliasis in goats has not been the subject
* Correspondingauthor. 0304-4017/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0304-4017(96)01019-9
172
J. V. Mbuh. B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
of much study. Furthermore, it is known that the course of fascioliasis in the host varies according to the ruminant species. For example, fascioliasis is often chronic and subclinical in cattle, but acute and accompanied by high mortality in sheep (Soulsby, 1982). Furthermore, the immunological consequences of fascioliasis vary among different ruminant species. While cattle acquire some degree of resistance following a primary infection (Ross, 1966; Boray, 1969; Doyle, 1971; Doyle, 1973), sheep seem to remain highly susceptible to fascioliasis after previous infections (Sinclair, 1975; Knight, 1980; Sandeman and Howell, 1981). The situation in goats is not known. The differences in the severity of fascioliasis and immunological responses among different ruminant species was considered to be partly attributable to variations in time-course responsiveness of these hosts to different antigenic determinants of the parasite. Profound antigenic alterations are associated with the development of the worm from a juvenile fluke to egg-laying adult (Bennet and Threadgold, 1975; Rajasekariah and Howell, 1978; Hanna, 1980a; Hanna, 1980b; Sandeman and Howell, 1981; Bennet et al., 1982). This implies a corresponding variation in the immunological reaction of the host in the course of an infection. The time-course analysis of antibody response in sheep infected with F. gigantica has been studied recently (Guobadia and Fagbemi, 1995). In this paper, we have investigated the sequence of antibody response and the profile of circulating antigen in goats infected with F. gigantica by immunoblotting and ELISA before and after chemotherapy. In addition, we have identified the antigens which have potential usefulness in immunodiagnosis of the fluke infection in goats.
2. Materials and methods
2.1. Preparation of antigen Live, adult F. gigantica, Paramphistomum microbothrium and Dicrocoelium hospes were collected from goats slaughtered in a local abattoir. They were washed four times with 0.01 M phosphate buffered saline (PBS) pH 7.2 to remove all traces of bile and blood. Whole worm antigen of F. gigantica was prepared by homogenizing the worms (1 worm ml- ~ PBS) in PBS which was supplemented wih 0.25 mg ml- ~ leupeptin in a Ten Broeck tissue grinder in an ice bath. The leupeptin served as a protease inhibitor. The mixture was centrifuged at 10000 g for 20 min. The supernatants were stored at - 2 0 ° C until used. Whole worm antigen extracts of P. microbothrium (PmWWE) and D. hospes (DhWWE) were prepared in the same way. The protein concentrations were determined by the procedure described by Bradford (1976).
2.2. Experimental animals Six, 6-month-old goats which were rared under Fasciola free conditions and ascertained free of infection by coprological examination were used. Four of the goats were infected orally with 350 metacercariae of F. gigantica each. Two goats served as uninfected controls. Blood samples were collected at weekly intervals from the jugular vein of infected
J.V. Mbuh, B.O. Fagbemi/ Veterinary Parasitology 66 (1996) 171-179
173
goats for 12 weeks. On the 12th week the goats were treated with 33 mg kg -~ oxyclozanide (Vermofas, Bimeda, Dublin) to terminate the infection. Thereafter, serum samples were collected for another 12 weeks. Serum samples from non-infected goats served as controls. A second group of goats were immunized with either PmWWE or DhWWE (10 mg antigen) in Freund's complete adjuvant. Booster doses containing 5 mg antigen in Freund's incomplete adjuvant were given three times at weekly intervals. Serum samples were collected one week after the last immunization. All samples were stored at - 20°C. Faecal samples of infected animals were examined at weekly intervals for the presence of F. gigantica eggs by the sedimentation method of Thienpont et al. (1979). Three rabbits were each infected orally with 15 metacercariae of F. gigantica. Blood was collected after 14 weeks from the ear vein.
2.3. Enzyme-linked immunoelectrotransfer blot (EITB) The method used was that described by Tsang et al. (1983) and Santiago and Hillyer (1986). Antigens were prepared at 4 ml- 1 in a sample buffer of 0.5 M Tris-HC1, 10% glycerol, 10% sodium dodecyl sulphate, 5% B-mercaptoethanol and 0.1% bromophenol blue. Whole worm extracts of F. gigantica antigens were electrophoresed in 12.5% polyacrylamide gel at 150 volts for 1½ h in a protein 11 minigel apparatus (BIORAD). Transfer of proteins from gel to nitrocellulose sheets was performed at 30 volts overnight at 4°C. The nitrocellulose sheet was cut into strips and incubated with the test sera which were diluted 1:200 with 0.05% PBS-Tween 20. The test sera consisted of those from goats with experimental F. gigantica infection, anti-PmWWE and antiDhWWE. This was followed by incubation in a 1:500 dilution of a horseradish peroxidase conjugated rabbit anti-goat IgG in 0.3% PBS-Tween 20. The strips were developed by the addition of 3, 3'-diaminobenzidine tetrahydrochloride (Sigma).
2.4. Enzyme linked immunosorbent assay (ELISA) ELISA was done as described by Santiago et al. (1986). Optimum working dilution of antigen as determined by checkerboard titration was 5 mg ml -I. The antigen was diluted in carbonate buffer pH 9.3 and incubated overnight at 4°C. Test sera were used at 1:200 dilution in 1% skimmed milk in 0.3% PBS-Tween 20. This was followed by incubation for 2 h at 37~C. Horseradish peroxidase conjugated anti-goat IgG (Kirkegaard and Perry Laboratories, Gaithensburg, MD) at a dilution of 1:1000 in 0.3% P B S / T w e e n 20 was then incubated for 1 h. O-phenylene diamine was used as substrate and incubated for 30 min at room temperature in the dark. A solution of 0.25 M H2SO 4 was added (50 I~1 per well) to terminate the reaction. Absorbance values were read spectrophotometrically at 492 nm.
2.5. Conjugation of rabbit anti-Fasciola serum to horseradish peroxidase Sera obtained from rabbits infected with F. gigantica was conjugated to horseradish peroxidase by the glutaraldehyde method (Lunde et al., 1987).
174
J. V. Mbuh, B.O. Fag bemi / Veterinary Parasitology 66 (1996) 171-179
Briefly, 10 ml of rabbit anti-F, gigantica antiserum was added to 2.5 g ammonium sulphate for 20 h at room temperature. The mixture was centrifuged at 10000 g for 1 h at 4°C. The immunoglobulin precipitate was washed three times with 25 ml of 1.75 M ammonium sulphate and centrifuged three times. One ml of distilled water was added to the precipitate and it was dialysed at 4°C for 24 h against 0.050 M sodium acetate/0.021 M acetic acid pH 5.0, and finally against 0.1 M sodium chloride/sodium azide for 24 h. A total of 125 txl of glutaraldehyde was added to 10 ml of PBS. Horseradish peroxidase (0.01 g) was added to 200 ill of the glutaraldehyde mixture and left at room temperature. One ml of 0.1 M carbonate buffer (pH 7.2) was added to the glutaraldehyde mixture. This was dialysed against carbonate buffer (pH 9.2), changing twice in 4 h. Five mg of the F. gigantica immunoglobulin was added to the glutaraldehyde mixture containing peroxidase; 0.1 ml of 0.2 M lysine was then added, together with an equal volume of glycerol. This preparation was stored at 20°C until used.
2.6. Detection of circulating F. gigantica antigen using the direct ELISA method A total of 100 I~1 of goat sera from experimentally infected goats were plated in each well of ELISA plates overnight at 4°C. The plates were washed five times at 15 min intervals with 0.05% PBS-Tween 20, after which the unreacted binding sites were blocked by incubating the plates with 1% skimmed milk in 0.3% PBS Tween 20 for 1 h at 37°C. Incubation with peroxidase labelled rabbit anti-Fasciola IgG (1:1000 dilution) was for lh. After five washes of 15 min each, 100 i~1 of the substrate, O-phenylene-diamine (OPD) was added and incubated for 30 min in the dark for colour development. The reaction was stopped by adding 50 I~1 of 1 M H2SO 4 to each well. The absorbance was read spectrophotometrically at 492 nm. The cut-off point to define a positive or negative serum sample was established as the mean absorbance of the normal sera + 2 SD.
POST INFECTION 2 4 681012
POST TREATMENT 2 4 6 8 1 0 1 2 (:
90-7g-5g42352g14Fig. 1. Immunoblot of F. gigantica somatic antigen with sera of infected goats before and after chemotherapy. The sera of six goats were pooled each week for the test.
J.V. Mbuh, B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
F
O
175
p
95---62 55 3028
-
21,----14-----
Fig. 2. Comparative immunobiot of F. gigantica somatic antigen with goat anti-Dicrocoelium (D), anti-Fasciola (F) and anti-Paramphistomum (P) sera. The anti-F sera were pooled from goats with 10 week old F. gigantica infection.
1.0CHEMOTHERAPY
0.8
0.6
0.4
o_~__ < 0.2-
0.0 3
5'
7'
9"
~ 11
' 13
~ 15
I ~7
~ 19
~ 21
2 ~3
' 25
Time (weeks)
Fig. 3. ELISA absorbance values for sera of F. gigantica infected goats before and after chemotherapy with oxyclozanide. Horizontal broken line indicates mean absorbance of normal sera + 2SD = 0.25.
J.V. Mbuh, B.O. Fagbemi/ VeterinaryParasitology66 (1996) 171-179
176 3. Results
Fasciola eggs were detected in the faeces of the infected goats 10 weeks after infection and they were not detected 1 week after chemotherapy. In EITB, three polypeptides of molecular weight 80 kDa, 59 kDa and 44 kDa were recognized as early as 2 weeks after infection (Fig. 1). By the 4th week, more polypeptide bands ranging in molecular weight from 17 kDa to 90 kDa were recognized. The 29 kDa, 35 kDa and 59 kDa polypeptides were more intensely recognized as from the 6th week. The recognition of these protein bands persisted until the 12th week after infection. By 4 weeks after chemotherapy, recognition of most of the protein bands had ceased although faint bands were still noticed at Week 6. By 12 weeks the bands that could be seen were diffused and non distinctive and no definite banding pattern was observed. A comparative immunoblotting study of F. gigantica whole worm extract with anti-P, microbothrium, anti-D, hospes and anti-F, gigantica revealed cross-reactivities. Anti-D. hospes serum recognized bands between 55-90 kDa, while anti-P, microbothrium serum recognized protein bands of molecular weight between 44-90 kDa and anti-F, gigantica serum recognized bands ranging between 14-92 kDa. The bands that appeared to be specific to F. gigantica were 30 kDa, 28 kDa, 21 kDa, 17 kDa and 14 kDa (Fig. 2).
CHE M O T H E R A P Y
(I.5
E
]
3
5
7
9
11
13
15
17
19
21
23
25
']'ir,',e ( w e e k s )
Fig. 4, ELISAresults of circulating F. gigantica antigens of experimentally infected goals before and after chemotherapy. Horizontal broken line indicates mean absorbance of normal sera + 2SD = 0.15. Each point represents the mean of three replicates.
J.V. Mbuh, B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
177
ELISA showed a steady antibody increase as from 2 weeks post infection until Week 12 when an absorbance of 0.8 was obtained (Fig. 3). After chemotherapy, antibody levels started to fall as from Week 13. There was a sharp decline between Weeks 15 and 16, followed by a steady fall in absorbance until Week 24. In the antibody-detection assay, circulating Fasciola antigens were detected by Week l after infection. The peak absorbance of these antigens was detected from Week 6 after infection to Week 12 when chemotherapy was done. At 4 Weeks after chemotherapy, no circulating antigen was detected till the end of the experiment (Fig. 4).
4. Discussion
In this study, the antibody and circulating antigen profiles were studied by EITB and ELISA after infecion of goats with F. gigantica. The detectable antibody response observed 2 weeks after infection in ELISA is consistent with the trend observed in sheep infected with the flukes (Guobadia and Fagbemi, 1995). Moreover, the rapid decline in antibody level after chemotherapy falls in line with the observation in sheep. In this way, the patterns of antibody response to F. gigantica infection in goats and sheep are similar to that which was reported in sheep infected with F. hepatica (Zimmerman et al., 1982; Santiago and Hillyer, 1988). However, in immunoblotting studies, while there was a gradual increase in the number of polypeptide bands of F. gigantica recognized by infected sheep sera in the weeks after infection (Guobadia and Fagbemi, 1995), the number of antigen bands recognized by infected goat sera appeared to reach a maximum 4 weeks after infection. There was a rapid immune recognition of all the components of the complete antigen repertoire of F. gigantica by infected goat sera within 4 weeks after infection. Thereafter, differences in recognition was only a matter of intensity. This contrasts sharply with what was observed in sheep in which the 87 kDa antigen was recognized as late as 12 weeks after infection (Guobadia and Fagbemi, 1995). The rapid immune recognition of F. gigantica antigens by goats may have much significance in determining the course of the infection and in defining the degree of immunity in this species. Furthermore, the early recognition of these antigens increases their usefulness for immunodiagnosis in the early stage of infection. Comparative immunoblotting with anti-P, microbothrium and anti-D, hospes sera revealed that at least four antigens including the 17 kDa and 21 kDa seem to be specific to F. gigantica. Polypeptide antigens of the same 17 and 21 kDa molecular weights have been reported to be specific to F. gigantica in sheep (Guobadia and Fagbemi, 1995). These antigens are therefore potentially good immunodiagnostic antigens. However, it should be realized that immunization of goats with whole worm antigen of P. microbothrium and D. hospes may not produce the real representation of the normal immunological pattern that may be obtained after the infection of the animals by these trematodes. Circulating antigens normally show active and present infections. The disappearance of antigens 4 weeks after treatment is a good indication that the test is highly sensitive and will be a good immunodiagnostic assay. While in goats the antigens disappeared 4
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J. V. Mbuh, B.O. Fagbemi / Veterinary Parasitology 66 (1996) 171-179
weeks post treatment, in sheep these disappeared 3 weeks after treatment (Guobadia and Fagbemi, 1996). The immunological recognition of the polypeptide antigen of F. gigantica, especially the specific 17 KDa and 21 KDa antigens, by infected goat sera, occurs as early as 4 weeks after infection while coproiogical diagnosis is possible only after 10 weeks of infection. Since an assay based on the detection of these specific antigens will provide an attractive alternative to the conventional diagnosis of F. gigantica in goats and sheep, work is in progress in our laboratory to purify and use the 17 KDa and 21 KDa antigens for this purpose.
Acknowledgements This study was supported by Grant No B / I 981-1 from the International Foundation for Sciences, Sweden.
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