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IgG isotype specific immune response in rats infected with Fasciola hepatica
Veterinary Parasitology 79 (1998) 229±237
IgG isotype specific immune response in rats infected with Fasciola hepatica A. Paz, R. SaÂnchez-Andrade, R. Panadero, P. DõÂez-BanÄos*, P. Morrondo Parasitology and Parasitic Diseases, Department of Animal Pathology, Veterinary Faculty, Santiago de Compostela University, 27071-Lugo, Spain Received 26 January 1998; accepted 4 April 1998
Abstract Antibody responses (IgG1, IgG2a, IgG2b, and IgG2c subclasses) against Fasciola hepatica L. in rats infected with metacercariae were analysed by ELISA. Animals of group 1 (R-1) remained infected throughout 21 weeks, and rats of group 2 (R-2) received a single oral dose of triclabendazole (Fasinex1 10%, Ciba-Geigy) at 4 weeks after infection. Group C (R-C) consisted of rats left intact which served as uninfected controls. All IgG subclasses increased during the first weeks after infection, but when triclabendazole was administered IgG1 and IgG2b diminished markedly. IgG2c showed a different profile to the other antibodies evaluated, increasing earliest and decreasing profoundly from the 9±11th week after infection (w.a.i.). The infected±untreated rats produced higher titres of antibodies than the rats of R-2, and these differences were statistically significant (p<0.05) in all subclasses evaluated except in IgG2a titres. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Fasciola hepatica; IgG isotype; ELISA; Rat; Triclabendazole
1. Introduction Infection of domestic livestock and experimental animals with Fasciola hepatica has been shown to induce an immunological response characterised by an increase in the levels of serum immunoglobulins, especially IgG. By means of ELISA tests, it has been proved that IgG titres remain elevated in serum throughout the infection, even long periods after chemoprophylaxis (MarõÂn, 1992; RodrõÂguez PeÂrez and Hillyer, 1995). This * Corresponding author. Tel.: +34 982 252231; fax: +34 982 252195; e-mail: [email protected] 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 6 5 - 4
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fact implies that during fasciolosis the release of antigenic products is prolonged, stimulating an important immune response (Poitou et al., 1993). Investigations have analysed the kinetics of IgG subclasses in parasitic infections (Horta and Ramalho-Pinto, 1984; Capron et al., 1987; Pearce et al., 1991; Smith et al., 1995), pointing that different responses occur during infection, and suggested that these observations implicate the stimulation of T-helper clones, classified as Th1 or Th2 on the basis of functional assays and secretion of lymphokines (Mosmann and Coffman, 1989). Nevertheless, although there are investigations in schistosomosis or filariosis, few studies have been carried out in rat fasciolosis, and none examined the kinetics of IgG2b and IgG2c levels. The aim of our study was to increase the understanding of the humoral immune responses manifested during the course of fasciolosis in rats. We have also tried to investigate any possible difference in the kinetics of these immunoglobulins that might be responsible for the long persistence of IgG titres in sera of infected animals, even after administration of a fasciolicide. To achieve these goals, we assessed the antibody response in more detail by analysing levels of different isotypes (IgG1, IgG2a, IGg2b and IgG2c) in two groups of rats infected with metacercariae of F. hepatica. We also evaluated the serum activity of the hepatic enzymes GLDH and g-GT, to establish the existence of pathological damage as a result of controlled infection with metacercariae of F. hepatica. 2. Material and methods Sprague±Dawley female rats were used in this trial as hosts for F. hepatica infection. Metacercariae of the trematode were obtained after infection of Lymnaea truncatula snails with F. hepatica miracidia, from eggs collected from livers of slaughtered cows. The viability of metacercariae was estimated in vitro, prior to infection, by microscopical examination (Paz Silva, 1997). Each rat received, by gastric tubing, 20 metacercariae. Animals were divided into three groups of nine rats in each one. Groups 1 (R-1) and 2 (R-2) were infected when 4 weeks old (80±120 g), and group C (R-C) was left as uninfected control. The rats of R-2 received a single dose of 10% triclabendazole (Fasinex1, Ciba-Geigy; 12 mg kgÿ1 b.w.), at 4 weeks after-infection (w.a.i.). All rats were bled weekly, from one week prior to infection to the 21st (w.a.i.) GLDH and g-GT levels were assayed using commercial kits (Merck1 and results are expressed in International Unit per litre (IU lÿ1). Systemic antibody responses were established by detecting IgG against the excretory/secretory (ES) antigen from F. hepatica adults using an indirect-ELISA (Paz Silva et al., 1997). Wells of micro-ELISA plates were coated with 100 ml ES (3.5 mg protein mlÿ1) and incubated for 3 h at 378C. After two washes, blocking of excess-binding sites was performed by incubation with 300 ml of PBS containing 0.05% Tween 20 and 1% skimmed milk (PTM) for 2 h at 378C. After two washes, rat sera diluted (1/20) in PTM were added in duplicate to the wells and incubated for 1 h at 378C. After 6 washes, 100 ml of horseradish peroxidase-conjugated (HRP-conjugated) rabbit anti-rat IgG1, IgG2a, IgG2b and IgG2c peroxidase conjugate (H&L, Nordic Immunological Laboratories, The Netherlands) diluted to 1:3000, 1:2000, 1:2000 and 1:2000 in PTM, respectively, were
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added and incubated at 378C for 1 h, according to Paz Silva (1997). After this, plates were washed four times with PBS containing 0.05% Tween 20 and two times with distilled water, and 100 ml of the substrate solution, made of 10 mg o-phenylenediamine (Sigma), 10 ml of 30% H2O2 in 12 ml of 0.1 M citrate buffer pH 4.5 was added and incubated for 10 min at room temperature in the dark. The reaction was stopped by adding 100 ml of 3N H2SO4, and absorbance determined at 450 nm in a microplate reader. Statistical analysis was performed using the non-parametric Kruskal±Wallis test for a global comparison, and when differences were significant, the non-parametric Mann± Whitney test for comparisons among two groups was used. 3. Results All rats of R-1 harboured flukes at necropsy and F. hepatica eggs appeared in their faecal samples at the 7th w.a.i. However, we did not find any worms in the liver of the rats of R-2 and none of these animals had a positive egg count. In R-1 GLDH levels increased significantly from the 3rd w.a.i. and remained higher than in the control group (R-C) until the 7th w.a.i. (Table 1). R-2 also rose from the 3rd w.a.i., but decreased from the 4th w.a.i., when triclabendazole was administered. g-GT levels were only detected in the infected±untreated rats (R-1) (Table 2), from the 5th w.a.i., reached one peak at the 9th w.a.i. and decreased until the end of the study, maintaining high values. In the treated and control groups this enzyme activity was not detected. Statistical analysis showed significant differences between R-1 and R-2 both in GLDH and g-GT levels after treatment (p<0.05). The IgG1 subclass began to increase at the 1st w.a.i. in both infected groups (Fig. 1(a)), and optical density values were significantly higher than in controls ( 2117.5704;
Table 1 Serum values of the glutamate dehydrogenase enzyme activity. Results are meansS.E.M., n8 rats. R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatement; R-1: rats infected which remained without treatment; R-C: uninfected rats which served as controls Weeks after infection
Group R-1 (IU lÿ1)
Group R-2 (IU lÿ1)
Group R-C (IU lÿ1)
0 1 3 5 7 9 11 13 15 17 19 21
0.56410.1021 2.78771.0823 11.91392.0673 11.47891.1208 3.67451.4909 0.84430.4049 0.63950.2165 0.74660.2739 0.71080.2334 0.64250.2568 0.68930.2144 0.64500.1921
0.54230.1207 3.06500.8898 12.98092.2742 27.45643.3809 8.17820.8401 0.95360.4115 1.12610.2198 1.07460.2481 1.01850.2696 1.04470.2364 1.40160.2121 1.39230.1938
0.62120.1061 1.14560.1132 1.27130.1098 1.53410.0987 1.45760.1224 1.35580.1123 1.33240.1071 1.29980.1625 1.25630.1614 1.26740.1187 1.16010.0874 1.14590.0993
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Table 2 Serum values of the g-glutamyl-transferase enzyme activity. Results are meansS.E.M., n8 rats. R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatment; R-1: rats infected which remained without treatment; R-C: uninfected rats which served as controls Weeks after infection
Group R-1 (IU lÿ1)
Group R-2 (IU lÿ1)
Group R-C (IU lÿ1)
0 1 3 5 7 9 11 13 15 17 19 21
0.51270.1763 0.49050.2189 0.52540.1703 0.54970.2820 0.58120.2394 0.58340.2822 0.48750.1497 0.70860.1563 0.56430.2395 0.58380.1943 0.62000.2014 0.76860.1662
0.50480.1528 0.52120.2970 0.50500.3132 1.37210.4099 24.04912.4461 31.02731.9656 21.090302.9469 5.50451.5890 2.45290.7405 3.24360.8517 2.71000.7013 2.38070.9202
0.52210.1768 0.52320.2317 0.53240.1879 0.62380.2653 0.57720.2481 0.49010.1723 0.51090.1845 0.50680.1843 0.48450.2291 0.47810.1957 0.46620.2312 0.41970.2105
p0.0000). The absorbances gradually rose throughout infection in R-1, whereas in R-2 increased to 7th w.a.i. (3 weeks after treatment) and reached a constant value till the end of the study. Using the Mann±Whitney test we assessed that these differences were significant (Uÿ5.8469; z0.0000). The IgG2a titres also began to rise from the 1st w.a.i. in R-1 and R-2 (Fig. 1(b)), and reached significantly higher values than in R-C 2111.2554; p0.0000). Nevertheless, IgG2a absorbances did not decrease until the 13th w.a.i. in R-2, remaining similar to that of R-1. These differences were not significant (p>0.05). As occurred in IgG1 levels, IgG2b increased from the 1st w.a.i. in both infected groups (Fig. 1(c)); at 5 w.a.i. (1 week after treatment) absorbance values decreased in R-2 to the 9 w.a.i., remaining constant to the end of the experience. In the infected and non-treated rats (R-1), IgG2b response dropped from the 11th w.a.i., but maintained higher values than the treated and control groups till the end of the study. The IgG2c titres were different to the IgG1, IgG2a and IgG2b, since a plateau was achieved in R-1 from the 1st w.a.i. to the 9 w.a.i., when it began to decrease until the end of the study (Fig. 1(d)). In R-2, absorbances dropped from the 5th w.a.i. This IgG2c isotype was the unique that did not increase during the course of infection. After chemotherapy with triclabendazole (4th w.a.i.), the most important features of the response of the immunoglobulin isotypes were a significant reduction in the titres of the IgG2b and especially in IgG2c response. Nevertheless, IgG1 and IgG2a absorbances maintained high values after treatment, showing a little reduction at the end of the trial. We observed that IgG2b and IgG2c titres dropped 1 week after treatment and IgG1 5 weeks after. Likewise, the absorbances of these three IgG isotypes were below the values of the infected±untreated rats (R-1) from the 4th w.a.i. Finally, the titres of IgG2a were very similar to those of the rats of R-1, decreasing moderately from the 13th w.a.i.
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Fig. 1. Immunoglobulin (IgG) isotype responses to F. hepatica excretory/secretory antigen. Results are meansS.E.M., n8 rats. (a) IgG1; (b) IgG2a; (c) IgG2b; (d) IgG2c. () R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatment; (&) R-1: rats infected which remained without treatment; () R-C: uninfected rats which served as controls.
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Fig. 1. (Continued)
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4. Discussion This is the first work in which the analysis of the subclasses of IgG in rats infected with metacercariae of F. hepatica and treated with triclabendazole was carried out. All infected rats achieved high levels of anti-Fasciola antibodies throughout the infection, and analysis of the isotypes of IgG involved in the rat humoral response revealed that each isotype showed remarkable independence in its response to different life cycle stages. The kinetics of the IgG1 and IgG2a responses in the remaining infected rats (R-1) was similar to those observed by Poitou et al. (1993), raising above control values by the first weeks after infection. The serum levels of IgG2b and IgG2c (not measured by Poitou et al., 1993) also increased during the 1st w.a.i. We appreciated that all IgG subclasses evaluated rose when flukes were migrating through the intestinal wall, peritoneo and liver parenchyma, as reflected by a significant increment in GLDH activity. Once flukes reached the bile ducts and attained maturity, evidenced by the highest values in g-GT maturity from 5±9th w.a.i. and the appearance of eggs in the faeces of the rats of R-1, IgG2b and IgG2c titres dropped up to 21st w.a.i. The response of IgG2b and IgG2c increased more quickly than IgG2a and IgG1. These results could indicate that stage specific antigenic determinants elicit strong responses from a particular isotype, as reported by Khalife et al. (1985) in rat schisotosmosis. It is particularly to be emphasised that the IgG1 and IgG2a antibodies to F. hepatica did not decrease since continued to rise over infection. These results seem to support the hypothesis that, during its development, F. hepatica releases substances that stimulate the rat lymphocyte response to the production of various subclasses of IgG. Decrease in the titres of IgG2b and IgG2c could be explained because a specific suppression of selected isotypes by a factor induced by F. hepatica might be produced, or because the IgG2b and IgG2c antibodies might be binding to circulating antigens, as suggested by Lawrence and Denham (1993). Another possibility could be that the production of IgG2b and IgG2c was markedly stimulated when flukes migrate through intestinal wall, peritoneum and liver parenchyma. To investigate whether some of these hypotheses could be clarified, we treated one group of infected rats with a single dose of triclabendazole (at 4 w.a.i.). No worms appeared in the bile ducts of the treated animals, GLDH levels declined 1 week after treatment, and g-GT activity was not detected, as occurred in the control group. The kinetics of the subclasses of IgG of R-1 and R-2 neither showed an identical pattern. IgG2b and IgG2c titres decreased as early as 1 week after treatment, but the values of IgG1 and IgG2a antibodies did not decrease until 5±7 weeks after the administration of the fasciolicide. This appears to indicate that the production of IgG2b and IgG2c antibodies is strikingly related to the passage and/or presence of F. hepatica in the liver parenchyma. So, when worms left the liver parenchyma and established in the bile ducts (R-1) or were eliminated by the action of triclabendazole (R-2), the stimulus to the production of IgG2b and IgG2c antibodies ended, and the titres of these subclasses dropped. Meeusen and Brandon (1994) pointed that while serum antibodies remained high after infection and cure, no antibody secreting cell (ASC) responses were detected in lymph nodes draining the target organs, unless the parasite were present in the tissue at the time of examination. These authors suggested that antibody levels in the serum were
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maintained by ASC located outside the lymph nodes, possibly in the bone marrow. It seems very conceivable that during the infection of rats with F. hepatica two different responses took place. First, the secretion of IgG2b and IgG2c is enhanced, being replaced by the production of IgG1 and IgG2a antibodies afterwards. These different responses could concern the involvement of Th1 and Th2-like cells, and it seems very probable that the Th2 phenotype is profoundly increased over infection, whereas the Th1 phenotype is either suppressed or possibly rendered anergic. It is interesting to note that in mice, the association of IgG2a, IgG2b and IgG3 antibodies to Th1 cells, and IgG1 antibodies to Th2 cells is well documented (Mosmann and Coffman, 1989). Lawrence and Denham (1993) reported that rat IgG1 and IgG2a are closely related to mouse IgG1, rat IgG2b is closely related to mouse IgG2a and IgG2b, and the rat IgG2c gene is most homologous to mouse IgG3. Finally, one of the main problems of ELISA-tests to evaluate the efficacy of a fasciolicide chemotherapy is the persistence of IgG antibodies for long periods, even after removal of flukes (MarõÂn, 1992). Taking account of our results, these disadvantages are due to IgG1 and IgG2a responses maintained high values until 5±7 weeks after treatment, whereas IgG2b and IgG2c reduced as early as 1 week after treatment. Acknowledgements This research was supported by the project PB95-0891-C02-01, from DGICYT (M.E.C., Spain). References Capron, A., Dessaint, J.P., Capron, M., Ouma, J.H., Butterworth, E.A., 1987. Immunity to schistosomes: Progress toward vaccine. Science 238, 1065±1072. Horta, M.F.M., Ramalho-Pinto, J.F., 1984. Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo. J. Immunol. 133, 3326±3332. Khalife, J., Capron, M., Grrzych, J.-M., Bazin, H., Capron, A., 1985. Fc-g receptors on rat eosinophils: Isotypedependent cell activation. J. Immunol. 135, 2780±2784. Lawrence, R.A., Denham, D.A., 1993. Stage and isotype specific immune responses in a rat model of filariasis. Paras. Immunol. 15, 429±439. MarõÂn, S., 1992. EpizootiologõÂa de la fasciolosis bovina en Asturias. IdentificacioÂn y expresioÂn de un antõÂgeno unitario. Tesis doctoral, Universidad de Oviedo, 133 pp. Meeusen, E., Brandon, M., 1994. The use of antibody-secreting cell probes to reveal tissue-restricted immune responses during infection. Eur. J. Immunol. 24, 469±474. Mosmann, T.R., Coffman, R.L., 1989. Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7, 145±173. Paz Silva, A., 1997. Estudio de las respuestas enzimaÂtica e inmunitaria en ratas infectadas con Fasciola hepatica: ValoracioÂn mediante las teÂcnicas inmunoenzimaÂticas ELISA e inmunoelectrotransferencia (EITB) en animales reinfectados y tratados. Tesis doctoral, Universidad de Santiago de Compostela University, 256 pp. Paz Silva, A., SaÂnchez-Andrade, R., Panadero FontaÂn, R., DõÂez BanÄos, P., Morrondo Pelayo, P., 1997. Histopathological changes and antibody response in rats infected with Fasciola hepatica and treated with triclabendazole. Helminthologia 34, 3±8.
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Pearce, E.J., Caspar, P., Grzych, J.M., Lewis, F.A., Sher, A., 1991. Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni. J. Experim. Med. 173, 159±166. Poitou, I., Baeza, E., Boulard, C., 1993. Kinetic responses of parasite-specific antibody isotypes, blood leucocyte pattern and lymphocyte subset in rats during primary infestation with Fasciola hepatica. Vet. Parasitol. 49, 179±190. RodrõÂguez PeÂrez, J., Hillyer, G.V., 1995. Detection of excretory±secretory circulating antigens in sheep infected with Fasciola hepatica and with Schistosoma mansoni and Fasciola hepatica. Vet. Parasitol. 56, 57±66. Smith, N.C., Ovington, K.S., Deplazes, P., Eckert, J., 1995. Cyotkine and immunoglobulin subclass responses of rats to infection with Eimeria nieschulzii. Parasitology 111, 51±57.
IgG isotype specific immune response in rats infected with Fasciola hepatica A. Paz, R. SaÂnchez-Andrade, R. Panadero, P. DõÂez-BanÄos*, P. Morrondo Parasitology and Parasitic Diseases, Department of Animal Pathology, Veterinary Faculty, Santiago de Compostela University, 27071-Lugo, Spain Received 26 January 1998; accepted 4 April 1998
Abstract Antibody responses (IgG1, IgG2a, IgG2b, and IgG2c subclasses) against Fasciola hepatica L. in rats infected with metacercariae were analysed by ELISA. Animals of group 1 (R-1) remained infected throughout 21 weeks, and rats of group 2 (R-2) received a single oral dose of triclabendazole (Fasinex1 10%, Ciba-Geigy) at 4 weeks after infection. Group C (R-C) consisted of rats left intact which served as uninfected controls. All IgG subclasses increased during the first weeks after infection, but when triclabendazole was administered IgG1 and IgG2b diminished markedly. IgG2c showed a different profile to the other antibodies evaluated, increasing earliest and decreasing profoundly from the 9±11th week after infection (w.a.i.). The infected±untreated rats produced higher titres of antibodies than the rats of R-2, and these differences were statistically significant (p<0.05) in all subclasses evaluated except in IgG2a titres. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Fasciola hepatica; IgG isotype; ELISA; Rat; Triclabendazole
1. Introduction Infection of domestic livestock and experimental animals with Fasciola hepatica has been shown to induce an immunological response characterised by an increase in the levels of serum immunoglobulins, especially IgG. By means of ELISA tests, it has been proved that IgG titres remain elevated in serum throughout the infection, even long periods after chemoprophylaxis (MarõÂn, 1992; RodrõÂguez PeÂrez and Hillyer, 1995). This * Corresponding author. Tel.: +34 982 252231; fax: +34 982 252195; e-mail: [email protected] 0304-4017/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 1 6 5 - 4
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fact implies that during fasciolosis the release of antigenic products is prolonged, stimulating an important immune response (Poitou et al., 1993). Investigations have analysed the kinetics of IgG subclasses in parasitic infections (Horta and Ramalho-Pinto, 1984; Capron et al., 1987; Pearce et al., 1991; Smith et al., 1995), pointing that different responses occur during infection, and suggested that these observations implicate the stimulation of T-helper clones, classified as Th1 or Th2 on the basis of functional assays and secretion of lymphokines (Mosmann and Coffman, 1989). Nevertheless, although there are investigations in schistosomosis or filariosis, few studies have been carried out in rat fasciolosis, and none examined the kinetics of IgG2b and IgG2c levels. The aim of our study was to increase the understanding of the humoral immune responses manifested during the course of fasciolosis in rats. We have also tried to investigate any possible difference in the kinetics of these immunoglobulins that might be responsible for the long persistence of IgG titres in sera of infected animals, even after administration of a fasciolicide. To achieve these goals, we assessed the antibody response in more detail by analysing levels of different isotypes (IgG1, IgG2a, IGg2b and IgG2c) in two groups of rats infected with metacercariae of F. hepatica. We also evaluated the serum activity of the hepatic enzymes GLDH and g-GT, to establish the existence of pathological damage as a result of controlled infection with metacercariae of F. hepatica. 2. Material and methods Sprague±Dawley female rats were used in this trial as hosts for F. hepatica infection. Metacercariae of the trematode were obtained after infection of Lymnaea truncatula snails with F. hepatica miracidia, from eggs collected from livers of slaughtered cows. The viability of metacercariae was estimated in vitro, prior to infection, by microscopical examination (Paz Silva, 1997). Each rat received, by gastric tubing, 20 metacercariae. Animals were divided into three groups of nine rats in each one. Groups 1 (R-1) and 2 (R-2) were infected when 4 weeks old (80±120 g), and group C (R-C) was left as uninfected control. The rats of R-2 received a single dose of 10% triclabendazole (Fasinex1, Ciba-Geigy; 12 mg kgÿ1 b.w.), at 4 weeks after-infection (w.a.i.). All rats were bled weekly, from one week prior to infection to the 21st (w.a.i.) GLDH and g-GT levels were assayed using commercial kits (Merck1 and results are expressed in International Unit per litre (IU lÿ1). Systemic antibody responses were established by detecting IgG against the excretory/secretory (ES) antigen from F. hepatica adults using an indirect-ELISA (Paz Silva et al., 1997). Wells of micro-ELISA plates were coated with 100 ml ES (3.5 mg protein mlÿ1) and incubated for 3 h at 378C. After two washes, blocking of excess-binding sites was performed by incubation with 300 ml of PBS containing 0.05% Tween 20 and 1% skimmed milk (PTM) for 2 h at 378C. After two washes, rat sera diluted (1/20) in PTM were added in duplicate to the wells and incubated for 1 h at 378C. After 6 washes, 100 ml of horseradish peroxidase-conjugated (HRP-conjugated) rabbit anti-rat IgG1, IgG2a, IgG2b and IgG2c peroxidase conjugate (H&L, Nordic Immunological Laboratories, The Netherlands) diluted to 1:3000, 1:2000, 1:2000 and 1:2000 in PTM, respectively, were
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added and incubated at 378C for 1 h, according to Paz Silva (1997). After this, plates were washed four times with PBS containing 0.05% Tween 20 and two times with distilled water, and 100 ml of the substrate solution, made of 10 mg o-phenylenediamine (Sigma), 10 ml of 30% H2O2 in 12 ml of 0.1 M citrate buffer pH 4.5 was added and incubated for 10 min at room temperature in the dark. The reaction was stopped by adding 100 ml of 3N H2SO4, and absorbance determined at 450 nm in a microplate reader. Statistical analysis was performed using the non-parametric Kruskal±Wallis test for a global comparison, and when differences were significant, the non-parametric Mann± Whitney test for comparisons among two groups was used. 3. Results All rats of R-1 harboured flukes at necropsy and F. hepatica eggs appeared in their faecal samples at the 7th w.a.i. However, we did not find any worms in the liver of the rats of R-2 and none of these animals had a positive egg count. In R-1 GLDH levels increased significantly from the 3rd w.a.i. and remained higher than in the control group (R-C) until the 7th w.a.i. (Table 1). R-2 also rose from the 3rd w.a.i., but decreased from the 4th w.a.i., when triclabendazole was administered. g-GT levels were only detected in the infected±untreated rats (R-1) (Table 2), from the 5th w.a.i., reached one peak at the 9th w.a.i. and decreased until the end of the study, maintaining high values. In the treated and control groups this enzyme activity was not detected. Statistical analysis showed significant differences between R-1 and R-2 both in GLDH and g-GT levels after treatment (p<0.05). The IgG1 subclass began to increase at the 1st w.a.i. in both infected groups (Fig. 1(a)), and optical density values were significantly higher than in controls ( 2117.5704;
Table 1 Serum values of the glutamate dehydrogenase enzyme activity. Results are meansS.E.M., n8 rats. R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatement; R-1: rats infected which remained without treatment; R-C: uninfected rats which served as controls Weeks after infection
Group R-1 (IU lÿ1)
Group R-2 (IU lÿ1)
Group R-C (IU lÿ1)
0 1 3 5 7 9 11 13 15 17 19 21
0.56410.1021 2.78771.0823 11.91392.0673 11.47891.1208 3.67451.4909 0.84430.4049 0.63950.2165 0.74660.2739 0.71080.2334 0.64250.2568 0.68930.2144 0.64500.1921
0.54230.1207 3.06500.8898 12.98092.2742 27.45643.3809 8.17820.8401 0.95360.4115 1.12610.2198 1.07460.2481 1.01850.2696 1.04470.2364 1.40160.2121 1.39230.1938
0.62120.1061 1.14560.1132 1.27130.1098 1.53410.0987 1.45760.1224 1.35580.1123 1.33240.1071 1.29980.1625 1.25630.1614 1.26740.1187 1.16010.0874 1.14590.0993
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Table 2 Serum values of the g-glutamyl-transferase enzyme activity. Results are meansS.E.M., n8 rats. R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatment; R-1: rats infected which remained without treatment; R-C: uninfected rats which served as controls Weeks after infection
Group R-1 (IU lÿ1)
Group R-2 (IU lÿ1)
Group R-C (IU lÿ1)
0 1 3 5 7 9 11 13 15 17 19 21
0.51270.1763 0.49050.2189 0.52540.1703 0.54970.2820 0.58120.2394 0.58340.2822 0.48750.1497 0.70860.1563 0.56430.2395 0.58380.1943 0.62000.2014 0.76860.1662
0.50480.1528 0.52120.2970 0.50500.3132 1.37210.4099 24.04912.4461 31.02731.9656 21.090302.9469 5.50451.5890 2.45290.7405 3.24360.8517 2.71000.7013 2.38070.9202
0.52210.1768 0.52320.2317 0.53240.1879 0.62380.2653 0.57720.2481 0.49010.1723 0.51090.1845 0.50680.1843 0.48450.2291 0.47810.1957 0.46620.2312 0.41970.2105
p0.0000). The absorbances gradually rose throughout infection in R-1, whereas in R-2 increased to 7th w.a.i. (3 weeks after treatment) and reached a constant value till the end of the study. Using the Mann±Whitney test we assessed that these differences were significant (Uÿ5.8469; z0.0000). The IgG2a titres also began to rise from the 1st w.a.i. in R-1 and R-2 (Fig. 1(b)), and reached significantly higher values than in R-C 2111.2554; p0.0000). Nevertheless, IgG2a absorbances did not decrease until the 13th w.a.i. in R-2, remaining similar to that of R-1. These differences were not significant (p>0.05). As occurred in IgG1 levels, IgG2b increased from the 1st w.a.i. in both infected groups (Fig. 1(c)); at 5 w.a.i. (1 week after treatment) absorbance values decreased in R-2 to the 9 w.a.i., remaining constant to the end of the experience. In the infected and non-treated rats (R-1), IgG2b response dropped from the 11th w.a.i., but maintained higher values than the treated and control groups till the end of the study. The IgG2c titres were different to the IgG1, IgG2a and IgG2b, since a plateau was achieved in R-1 from the 1st w.a.i. to the 9 w.a.i., when it began to decrease until the end of the study (Fig. 1(d)). In R-2, absorbances dropped from the 5th w.a.i. This IgG2c isotype was the unique that did not increase during the course of infection. After chemotherapy with triclabendazole (4th w.a.i.), the most important features of the response of the immunoglobulin isotypes were a significant reduction in the titres of the IgG2b and especially in IgG2c response. Nevertheless, IgG1 and IgG2a absorbances maintained high values after treatment, showing a little reduction at the end of the trial. We observed that IgG2b and IgG2c titres dropped 1 week after treatment and IgG1 5 weeks after. Likewise, the absorbances of these three IgG isotypes were below the values of the infected±untreated rats (R-1) from the 4th w.a.i. Finally, the titres of IgG2a were very similar to those of the rats of R-1, decreasing moderately from the 13th w.a.i.
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Fig. 1. Immunoglobulin (IgG) isotype responses to F. hepatica excretory/secretory antigen. Results are meansS.E.M., n8 rats. (a) IgG1; (b) IgG2a; (c) IgG2b; (d) IgG2c. () R-2: rats infected with metacercariae of F. hepatica and treated with a single dose of triclabendazole 4 weeks after treatment; (&) R-1: rats infected which remained without treatment; () R-C: uninfected rats which served as controls.
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Fig. 1. (Continued)
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4. Discussion This is the first work in which the analysis of the subclasses of IgG in rats infected with metacercariae of F. hepatica and treated with triclabendazole was carried out. All infected rats achieved high levels of anti-Fasciola antibodies throughout the infection, and analysis of the isotypes of IgG involved in the rat humoral response revealed that each isotype showed remarkable independence in its response to different life cycle stages. The kinetics of the IgG1 and IgG2a responses in the remaining infected rats (R-1) was similar to those observed by Poitou et al. (1993), raising above control values by the first weeks after infection. The serum levels of IgG2b and IgG2c (not measured by Poitou et al., 1993) also increased during the 1st w.a.i. We appreciated that all IgG subclasses evaluated rose when flukes were migrating through the intestinal wall, peritoneo and liver parenchyma, as reflected by a significant increment in GLDH activity. Once flukes reached the bile ducts and attained maturity, evidenced by the highest values in g-GT maturity from 5±9th w.a.i. and the appearance of eggs in the faeces of the rats of R-1, IgG2b and IgG2c titres dropped up to 21st w.a.i. The response of IgG2b and IgG2c increased more quickly than IgG2a and IgG1. These results could indicate that stage specific antigenic determinants elicit strong responses from a particular isotype, as reported by Khalife et al. (1985) in rat schisotosmosis. It is particularly to be emphasised that the IgG1 and IgG2a antibodies to F. hepatica did not decrease since continued to rise over infection. These results seem to support the hypothesis that, during its development, F. hepatica releases substances that stimulate the rat lymphocyte response to the production of various subclasses of IgG. Decrease in the titres of IgG2b and IgG2c could be explained because a specific suppression of selected isotypes by a factor induced by F. hepatica might be produced, or because the IgG2b and IgG2c antibodies might be binding to circulating antigens, as suggested by Lawrence and Denham (1993). Another possibility could be that the production of IgG2b and IgG2c was markedly stimulated when flukes migrate through intestinal wall, peritoneum and liver parenchyma. To investigate whether some of these hypotheses could be clarified, we treated one group of infected rats with a single dose of triclabendazole (at 4 w.a.i.). No worms appeared in the bile ducts of the treated animals, GLDH levels declined 1 week after treatment, and g-GT activity was not detected, as occurred in the control group. The kinetics of the subclasses of IgG of R-1 and R-2 neither showed an identical pattern. IgG2b and IgG2c titres decreased as early as 1 week after treatment, but the values of IgG1 and IgG2a antibodies did not decrease until 5±7 weeks after the administration of the fasciolicide. This appears to indicate that the production of IgG2b and IgG2c antibodies is strikingly related to the passage and/or presence of F. hepatica in the liver parenchyma. So, when worms left the liver parenchyma and established in the bile ducts (R-1) or were eliminated by the action of triclabendazole (R-2), the stimulus to the production of IgG2b and IgG2c antibodies ended, and the titres of these subclasses dropped. Meeusen and Brandon (1994) pointed that while serum antibodies remained high after infection and cure, no antibody secreting cell (ASC) responses were detected in lymph nodes draining the target organs, unless the parasite were present in the tissue at the time of examination. These authors suggested that antibody levels in the serum were
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maintained by ASC located outside the lymph nodes, possibly in the bone marrow. It seems very conceivable that during the infection of rats with F. hepatica two different responses took place. First, the secretion of IgG2b and IgG2c is enhanced, being replaced by the production of IgG1 and IgG2a antibodies afterwards. These different responses could concern the involvement of Th1 and Th2-like cells, and it seems very probable that the Th2 phenotype is profoundly increased over infection, whereas the Th1 phenotype is either suppressed or possibly rendered anergic. It is interesting to note that in mice, the association of IgG2a, IgG2b and IgG3 antibodies to Th1 cells, and IgG1 antibodies to Th2 cells is well documented (Mosmann and Coffman, 1989). Lawrence and Denham (1993) reported that rat IgG1 and IgG2a are closely related to mouse IgG1, rat IgG2b is closely related to mouse IgG2a and IgG2b, and the rat IgG2c gene is most homologous to mouse IgG3. Finally, one of the main problems of ELISA-tests to evaluate the efficacy of a fasciolicide chemotherapy is the persistence of IgG antibodies for long periods, even after removal of flukes (MarõÂn, 1992). Taking account of our results, these disadvantages are due to IgG1 and IgG2a responses maintained high values until 5±7 weeks after treatment, whereas IgG2b and IgG2c reduced as early as 1 week after treatment. Acknowledgements This research was supported by the project PB95-0891-C02-01, from DGICYT (M.E.C., Spain). References Capron, A., Dessaint, J.P., Capron, M., Ouma, J.H., Butterworth, E.A., 1987. Immunity to schistosomes: Progress toward vaccine. Science 238, 1065±1072. Horta, M.F.M., Ramalho-Pinto, J.F., 1984. Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo. J. Immunol. 133, 3326±3332. Khalife, J., Capron, M., Grrzych, J.-M., Bazin, H., Capron, A., 1985. Fc-g receptors on rat eosinophils: Isotypedependent cell activation. J. Immunol. 135, 2780±2784. Lawrence, R.A., Denham, D.A., 1993. Stage and isotype specific immune responses in a rat model of filariasis. Paras. Immunol. 15, 429±439. MarõÂn, S., 1992. EpizootiologõÂa de la fasciolosis bovina en Asturias. IdentificacioÂn y expresioÂn de un antõÂgeno unitario. Tesis doctoral, Universidad de Oviedo, 133 pp. Meeusen, E., Brandon, M., 1994. The use of antibody-secreting cell probes to reveal tissue-restricted immune responses during infection. Eur. J. Immunol. 24, 469±474. Mosmann, T.R., Coffman, R.L., 1989. Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7, 145±173. Paz Silva, A., 1997. Estudio de las respuestas enzimaÂtica e inmunitaria en ratas infectadas con Fasciola hepatica: ValoracioÂn mediante las teÂcnicas inmunoenzimaÂticas ELISA e inmunoelectrotransferencia (EITB) en animales reinfectados y tratados. Tesis doctoral, Universidad de Santiago de Compostela University, 256 pp. Paz Silva, A., SaÂnchez-Andrade, R., Panadero FontaÂn, R., DõÂez BanÄos, P., Morrondo Pelayo, P., 1997. Histopathological changes and antibody response in rats infected with Fasciola hepatica and treated with triclabendazole. Helminthologia 34, 3±8.
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