Parasitology International 59 (2010) 147–153
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Immune response of goats immunised with glutathione S-transferase and experimentally challenged with Fasciola hepatica L. Buffoni a, R. Zafra b, A. Pérez-Écija b, F.J. Martínez-Moreno a, E. Martínez-Galisteo c, T. Moreno a, J. Pérez b, A. Martínez-Moreno a,⁎ a b c
Animal Health Department (Parasitology), Faculty of Veterinary Medicine, University of Córdoba, Campus de Rabanales, Ctra. Madrid-Cádiz, km 396, 14014, Córdoba, Spain Anatomy and Comparative Pathology Department, Faculty of Veterinary Medicine, University of Córdoba, Campus de Rabanales, Ctra. Madrid-Cádiz, km 396, 14014, Córdoba, Spain Biochemistry and Molecular Biology Department, Edificio Severo Ochoa, University of Córdoba, Campus de Rabanales, Ctra. Madrid-Cádiz, km 396, 14014, Córdoba, Spain
a r t i c l e
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Article history: Received 19 June 2009 Received in revised form 14 December 2009 Accepted 16 December 2009 Available online 24 December 2009 Keywords: Fasciola hepatica Immunisation FhGST Goats
a b s t r a c t Glutathione S-transferase (FhGST) purified from Fasciola hepatica adult worms was used to immunise goats against F. hepatica in an experimental infection; the level of protection, in terms of fluke burden, faecal egg counts and hepatic damage was determined, as well as the humoral and cellular immune response elicited. Animals were allocated into three groups of six animals each: group 1 (immunised with FhGST and infected), group 2 (unimmunised and infected), and group 3 (unimmunised and uninfected). There was no significant reduction of fluke burden (9.3%) or faecal egg counts; hepatic damage was also similar in both infected groups. However, immunisation with FhGST induced the development of a well-defined immune response, characterized by the production of specific-FhGST antibodies as well as the appearance of circulating IL-4. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Fasciolosis caused by the digenetic trematode Fasciola hepatica is a major worldwide disease of ruminants and responsible for economic losses estimated at over US$ 3.2 billion per annum, in spite of efforts to control the disease [1]. It is also considered an emerging zoonosis, particularly in hyperendemic areas of South America and Asia [2]. Although goat infection is considered less prevalent than ovine or bovine fasciolosis, goats are very sensitive and both chronic and acute processes have been described with important economic losses [3]. The control of fasciolosis is mostly carried out by the use of anthelmintics, and triclabendazole (TCBZ) is the drug of choice for treating liver fluke infections. However, the appearance of resistance to TCBZ reported in many countries [4], the costs of treatment in endemic areas with high prevalence, mainly for developing countries, and the risk related to the presence of drug residues in food of animal origin, are fostering the development of immunological control strategies of the disease.
Abbreviations: AST, aspartate aminotransferase; FCA, Freund's complete adjuvant; FEC, faecal egg counts; FhESP, Fasciola hepatica excretory–secretory products; FhGST, Fasciola hepatica glutathione S-transferase; FIA, Freund's incomplete adjuvant; GGT, gamma glutamyl transferase; GST, glutathione S-transferase; IL-4, Interleukin 4; IFN-γ, gamma interferon; PI, post-infection; TCBZ, triclabendazole; IL-10, Interleukin 10; TGFβ, Transforming Growth Factor beta. ⁎ Corresponding author. Tel.: + 34 957218721; fax: + 34 957211067. E-mail address:
[email protected] (A. Martínez-Moreno). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.12.005
Immunological resistance to F. hepatica infection has been achieved by the use of different vaccine candidates, although this resistance seems to be clearly host-dependent. High levels of protection against infection with F. hepatica have been described in mice [5], rabbits [6], sheep [5,7], and cattle [8,9]. To date, few vaccine trials have been conducted in goats [10] and there is scarce information on the immunological resistance of this host [3]. Several bioactive molecules have been evaluated as vaccine candidates with promising results, such as the cathepsin L proteinases [7,8], the fatty acid binding proteins [5], and the enzymes glutathione tranferases (GSTs) [9,11]. GSTs comprise a family of isoenzymes responsible for cellular detoxification [12], metabolic reactions of secondary molecules of lipid peroxidation [13], and are present at high levels in helminths, particularly digeneans trematodes [14]. Different vaccine trials have been conducted with GST: in 1990, sheep were vaccinated with GST purified from F. hepatica, and a 78% reduction in mean worm burdens was obtained [11]. Later on, a significant 69% reduction in fluke burden in bovine experimentally infected with F. hepatica was observed [9]. In contrast, sheep immunised with GST purified from Fasciola gigantica showed no significant level of protection, although a reduction in the fluke burden between 21% and 32% was obtained [15]. These results showed a promising path for GST vaccination of cattle and sheep; however, the induction of immune protection by GST in caprine fasciolosis has not been evaluated. The aim of this work was to study the level of protection and the immune response in goats
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immunised with native glutathione S-transferase and experimentally infected with F. hepatica.
incubated for 2 min in a cuvette spectrophotometer, and the absorbance was measured at 340 nm. Samples with highest protein concentration and enzymatic activity were stored at −80 °C.
2. Materials and methods 2.1. Animals
2.4. Liver enzyme analysis
Eighteen Florida breed goats (males), aged 5 to 6 months, were randomly allocated over three groups of six animals and maintained for one month quarantine. During this period, animals were confirmed to be free of liver fluke infection by faecal egg analysis and anti-IgG specific against F. hepatica antigens by ELISA method. Goats were fed with hay concentrated ad libitum, and housed indoor.
Aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) levels were analyzed from plasma samples by using spectrophotometry and a specific protocol described by Biosystems Reagents & Instruments. The enzymes were measured at 340 and 405 nm for AST and GGT respectively, and results were expressed as international units per litre (IU/l).
2.2. Experimental design Animals were allocated into 3 groups of 6 animals. For primary immunisation, animals of group 1 were injected with 200 μg of FhGST in 200 μl of FCA (Freund's complete adjuvant). Ten days later animals were boosted with a second and third injection of 200 μg of FhGST in 200 μl of FIA (Freund's incomplete adjuvant) each, at intervals of 10 days. All immunisation doses were administered by subcutaneous route into the shoulder region. Ninety days after first immunisation dose, groups 1 and 2 were orally challenged with 200 metacercariae of F. hepatica within gelatine capsules. Animals of group 3 remained as uninfective control. During the first week after the experimental infection, one of the animals from group 2 died due to abdominal trauma with abomasal perforation, not related to F. hepatica infection, therefore group 2 was finally reduced to 5 animals. Faecal and blood samples were taken weekly from each animal. Blood was collected using 10 ml NH 170 IU vacutainers (BD vacutainers) and plasma was separated by centrifuging at 1.285 g for 15 min, and stored at −20 °C until analyzed. All animals were killed at 44 weeks of experiment (21 week post-infection) by intravenous injection of T61® (Intervet, Barcelona, Spain). The experimental design and protocols were approved by the Bioethics Committee of the University of Córdoba with reference number 7119.
2.5. Faecal eggs and parasite counts Faecal samples were collected weekly from each animal and a sedimentation method was performed for detecting egg presence. The liver and intact gallbladders were dissected at necropsy. Gallbladders were opened and carefully examined for the presence of flukes. In the liver, bile ducts were cut open with blunt scissors and the flukes were recovered with blunt forceps. The liver was then cut into small pieces for the collection of remaining flukes. All flukes were counted and measured.
2.6. Evaluation of hepatic damage At necropsy, tissue samples of the left and right hepatic lobes were fixed in 10% buffered formalin and embedded in paraffin wax. Four μm thick tissue sections were stained with haematoxylin–eosin and periodic acid Schiff for histopathological examination. Gross and histopathological hepatic changes were evaluated by two pathologists and ranged as none (−), moderate (+), severe (++) and very severe (+++).
2.7. Detection of antibodies 2.3. Parasite and parasite antigens F. hepatica metacercariae of bovine origin were obtained from the Central Veterinary Laboratory of Weybridge (UK) and stored at 4 °C until used. Excretory–secretory antigen (FhESP) was obtained as previously described by Martínez et al. [3]. Briefly, adult liver flukes were collected from infected goats, washed and incubated with physiological saline pH 7.0 at 37 °C for 8 h. The suspension was centrifuged at 4 °C and 5.145 g for 30 min to remove all particles. The supernatant containing FhESP products was collected and protein concentration was measured using BCA protein assay kit (Pierce). GST was isolated and purified from F. hepatica adult worms by affinity chromatography, as described by Sexton et al. [11]. Briefly, F. hepatica adult flukes were homogenized in 20 ml buffer containing 0.5% Triton X100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl pH 8.0, 2 mM PMSF and supplemented with 2 mM DTT (added before used). Cellular extract was centrifuged at 12,000 g for 30 min at 4 °C, supernatant was collected and filtered using membrane filters of 0.22 μm pore (Millipore). The solution was used for purification of GST on GSH-agarose beads (Sigma) [11]. Bounded material was eluted from the beads with 50 mM Tris–HCl pH 8.5, 1 mM EDTA, 10 mM GSH, and absorbance at 280 nm and electrophoresis using polyacrylamide gels (one-dimensional SDS-PAGE) [16], were performed for the presence of protein. A measurement of the enzymatic activity of the purified GST was realized as follows: 10 μl of sample (containing purified GST) was mixed with 940 μl of buffer T (100 mM NaH2PO4 pH 6.5 and 1 mM EDTA), 25 μl GSH 40 mM, and 25 μl CDNB 40 mM;
Humoral immune response was evaluated by detection of antibodies against purified GST (FhGST) and FhESP antigen using the ELISA method as previously described by Martínez et al. [3] with some modifications. Briefly, 96 well microtiter plates (Maxisorp, Nunc) were coated with 100 μl/well of FhGST and FhESP (2.5 μg/ml and 2 μg/ml, respectively) diluted in 0.05 M carbonate–bicarbonate buffer pH 9.6 and incubated overnight at 4 °C. After 5 washes with phosphate buffer saline (PBS) 0.05% Tween 20, 100 μl/well of blocking buffer containing 1% BSA diluted in PBS was incubated at 37 °C for 30 min. The wells were washed 5 times and 100 μl/well of goat plasma diluted in blocking buffer at 1:100 for microplates coated with FhGST and 1:400 for microplates coated with FhESP were added, and incubated at RT for 30 min. After washing, 100 μl/well of horseradish peroxidase conjugated rabbit anti-goat IgG (Sigma) diluted at 1:5000 in blocking buffer was incorporated and incubated at RT for 30 min. Wells were washed and 100 μl/well of the chromogen substrate OPD 0.04% (ortho-phenylenediamidine dihydrochloride, Sigma) and hydrogen peroxide 30% w/v diluted in phosphate-citrate buffer pH 5.0 was added and incubated at RT for 15 min for microplates coated with FhGST and 10 min for microplates coated with FhESP. Reaction was stopped by adding 100 μl/well of 1 M sulphuric acid and optical density was measured at 492 nm with a Ceres UV 900® spectrophotometer (Bio-Tek Instruments). Plasma pools from 10 experimentally infected goats (containing more than 100 liver flukes each) and from 10 uninfected and free of liver flukes goats were used as positive and negative controls, respectively. All samples were analyzed in duplicate.
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fluke burden reduction of 9.3% was detected in animals immunised with FhGST, although this reduction was not significant.
2.8. Detection of IL-4 A capture ELISA was developed at our laboratory for measuring goat IL-4 from plasma samples. ELISA microplates (Maxisorp, Nunc) were coated with 100 μl/well of polyclonal chicken anti-ovine IL-4 (Cytocen) diluted at 2 μg/ml in 0.05 M carbonate–bicarbonate buffer pH 9.6 and incubated at 37 °C for 2 h, and overnight at 4 °C. Plates were washed 5 times with phosphate buffer saline (PBS) 0.05% Tween 20 and blocked with 100 μl/well of blocking buffer containing 1% BSA diluted in PBS, at 37 °C for 30 min. After washing, 100 μl/well of goat plasma was added and incubated at 37 °C for 2 h. Wells were washed and 100 μl/well of monoclonal mouse anti-bovine IL-4 (Serotec) diluted at 1:100 in blocking buffer was added and incubated at 37 °C for 1 h. After washing 100 μl/well of the polyclonal rabbit anti-mouse IgG:HRP (secondary antibody, Serotec) diluted at 1:500 in blocking buffer was added and incubated at 37 °C for 30 min. Plates were washed and 100 μl/well of the chromogen substrate OPD 0.04% (ortho-phenylenediamidine dihydrochloride, Sigma) and hydrogen peroxide 30% w/v diluted in phosphate-citrate buffer pH 5.0 was added and incubated at RT for 10 min. Reaction was stopped by adding 100 μl/well of 1 M sulphuric acid and optical density was measured at 492 nm with a Ceres UV 900® spectrophotometer (BioTek Instruments). As positive control sample, recombinant bovine IL4 (Serotec) was used. 2.9. Detection of IFN-γ The monoclonal antibody-based sandwich enzyme immunoassay BOVIGAMTM kit (Bovine gamma interferon test, CSL Veterinary) was used for detection of goat IFN-γ from plasma samples according to manufacturer's instructions. 2.10. Statistical analysis Statistical significance was calculated by performing nonparametric analyses using the Mann–Whitney U test to compare vaccinated and control animals, P values of 0.05 or lower were considered statistically significant.
3.2. Liver enzymes production Plasma levels of AST and GGT are shown in Fig. 1A and B, respectively. During immunisation period, no significant changes were detected for all groups regarding production of both AST and GGT. After challenge, production of AST was very similar for immunised (group 1) and infected animals (group 2), showing a significant increase (P b 0.01) between weeks 4 and 15, and reaching the higher values at weeks 11 and 9 for groups 1 and 2, respectively. Plasma level of GGT was significantly elevated (P b 0.01) from week 8 until week 15 both in the immunised and infected groups, and no significant differences were detected between them along the infection period.
3.3. Hepatic lesions Gross hepatic lesions are shown in Fig. 2 and consisted of fibrous perihepatitis and tortuous whitish scars involving mainly the left hepatic lobe. In both groups, 2 animals presented mild, moderate and severe gross hepatic lesions. Moderate to severe dilation of bile ducts was found on the visceral hepatic surface in both infected groups, as well as moderate to severe dilation of the gall bladder. No hepatic lesions were detected in animals of the uninfected group 3. Histopathological changes in both infected groups showed high individual variability (Table 2). These changes consisted of portal fibrosis and chronic hepatic fibrous tracts containing numerous hemosiderin-laden macrophages. Granulomas with eosinophilic necrotic centre surrounded by multinucleate cells, macrophages and peripheral fibrosis were commonly observed. Bile ducts showed moderate to marked hyperplasia and moderate to abundant portal infiltration of lymphocytes and plasma cells, often arranged in lymphoid follicles with germinal centres. Infiltration of eosinophils in portal spaces was common, particularly in group 2, while globule leukocyte infiltration in bile ducts was more consistently observed in the immunised group than in the non-immunised group.
3. Results 3.4. Antibody response to FhGST 3.1. Parasitological results In both infected groups eggs appeared at 10 week post-infection (pi), slightly increasing until week 13 pi and showing a steady increase from that on. The higher egg counts (750 and 800 eggs per gram for groups 2 and 1, respectively) were recorded between 17 and 20 weeks pi. No eggs or fluke worms were detected in animals of the uninfected group 3. A total of 290 and 266 liver fluke worms were collected from infected groups 1 and 2, respectively. The mean fluke burden and mean length of flukes are presented in Table 1. The data showed no significant differences in both mean fluke burdens and mean length of flukes (P N 0.05) between infected groups, although fluke size was smaller in the immunised group than in the unimmunised group. A
Production of FhGST-specific IgG for each group is shown in Fig. 3A. All animals immunised with FhGST (group 1) developed an antibody response from the first week after the first immunisation dose, until the end of the experiment; reaching the highest level 4 weeks after the first injection, and showing a slight decreasing tendency later on. In respect of animals of group 2, no modification of antibody levels was observed during immunisation period (as expected), whereas an elevation of IgG was detected after week 6 of the experimental infection. Statistical analysis showed significant differences (P b 0.05) between values obtained at weeks 6, 8, 9, 10, 12 and 13 of the infection period, compared against values from animals of group 3.
3.5. Antibody response to FhESP Table 1 Worm burden in group 1 (immunised and infected) and group 2 (unimmunised and infected).
Group 1 Group 2
Mean fluke burden (per goat)
Infection dose (percentage)
Mean length of flukes (mm)
Fluke burden reduction (percentage)
48.3 53.2
24.1 26.6
19.4 ± 3.5 26.7 ± 2.8
9.3 –
A similar pattern of IgG production was observed either for immunised and infected animals (group 1) or infected animals (group 2). During immunisation period, no antibody response was detected in immunised animals, whereas FhESP-specific antibodies were significantly increased (P b 0.01) 2 weeks after the experimental infection until the end of experiment. Production of specific anti-FhESP IgG is shown in Fig. 3B.
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Fig. 1. A: Plasma levels of AST. During the course of the infection, mean values showed a steady increase in both immunised (group 1) and unimmunised (group 2) goats. B: Plasma levels of GGT. Enzyme values were significantly elevated (P b 0.01) in both immunised and unimmunised infected groups (1 and 2), indicating a clear bile duct damage.
Fig. 2. A (group 1): diaphragmatic aspect of the liver showing numerous whitish tortuous tracts located mainly in the left lobe. B (group 2): severe fibrous perihepatitis on the diaphragmatic surface of the liver. C (group 1): severe diffuse inflammatory infiltration composed mainly by eosinophils and macrophages with brown pigment. HE. Bar, 250 μm. D (group 2): granulomas against the parasite with the presence of giant cells causing deformity of hepatic parenchyma. HE. Bar, 250 μm.
L. Buffoni et al. / Parasitology International 59 (2010) 147–153 Table 2 Hepatic histopathological changes in immunised and infected goats (group 1) and unimmunised and infected goats (group 2). Goats Group 1 1 2 3 4 5 6 Group 2 1 2 3 4 5
PH
CT
LPI
G
E
+ +++ ++ + +++ +
+ ++ +++ + ++ +
+ ++ ++ + ++ +
− +++ ++ − +++ +
+ +++ + ++ +++ ++
+ + +++ ++ ++
+ + +++ +++ ++
+ ++ +++ ++ ++
− +++ +++ ++ −
+ + + ++ ++
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3.7. Plasma levels of IFN-gamma Plasma levels of IFN-γ are presented in Fig. 4B. Results indicate that experimental infection induced a slight increase of IFN-γ production in both groups 1 and 2, but this elevation was minimum and not statistically significant when compared to the negative control group (group 3) and to values obtained from positive control samples. No IFN-γ production was detected following immunisation with FhGST. 4. Discussion
PH: fibrous perihepatitis; CT: chronic tracts; LPI: infiltration of lymphocytes and plasma cells; G: granulomas; E: infiltration of eosinophils. −: None; +: moderate; ++: severe; +++: very severe.
3.6. Plasma levels of IL-4 A sudden and significant (P b 0.05) increase of IL-4 level 8 weeks after first injection was detected in all animals immunised with FhGST (group 1), followed by an marked decrease until 3 weeks postinfection. During the rest of the infection period no significant elevation of IL-4 was observed. Levels of IL-4 from the infected animals (group 2) were slightly elevated during the late stage of the infection, from week 9 until the end of the experiment. No significant variations of values from the uninfected animals (group 3) were detected during the experiment (Fig. 4A).
The parasitological and pathological results of the present study indicate that no protection was developed in the animals immunised with native FhGST following experimental infection. There was neither significant reduction in faecal egg output nor fluke burden or hepatic damage in the immunised group when compared with the control group. Therefore, our results suggest that combination of FhGST with FCA/FIA adjuvant is not a good vaccine candidate for those animals. FhGST has not previously been used for vaccine trials in goats, but it has been assayed in cattle and sheep with variable results. Sheep vaccinated with several doses of FhGST in FCA showed a 57% reduction in fluke burden [11], but in additional studies it was not possible to consistently induce a protective response despite using comparable vaccination protocols [17]. FhGST also induced significant (41–69%) levels of protection in cattle; protection was dependent on the choice of adjuvant, with the highest protection observed with GST in Quil A/Squalene Montanide 80® [9]. Based on the observation that heterologous immunity between Schistosoma and Fasciola spp. has been demonstrated, recombinant GST from Schistosoma mansoni in combination with different adjuvants (aluminium hydroxide, Quil A
Fig. 3. A: Production of antibodies (whole IgG) against FhGST. Results are expressed as optical density (OD) and each point represents the mean absorbance at 490 nm. B: Antibody response to FhESP products. Results show OD values detected during the course of the experiment.
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Fig. 4. A: IL-4 level in plasma samples. A significant (P b 0.05) increase of IL-4 was recorded 8 weeks after initial immunisation with FhGST. Recombinant bovine IL-4 (1:10 diluted) was used as positive control (values ranged from 0.6 to 1). B: Mean values (OD) of IFN-γ analyzed from plasma samples. No significant production of IFN-γ was observed during the experiment. Values obtained from negative and positive control samples ranged from 0.6 to 0.75 and from 3 to 3.3, respectively.
and FCA) was also used as vaccine candidate in cattle, but no significant protection against F. hepatica was obtained [18]. It is also noticeable that GST from F. gigantica has been used in vaccine trials against F. gigantica, and no significant protection was observed [15], despite the fact that this parasite is more susceptible to immune effector mechanisms than F. hepatica [19]. Notwithstanding, immunisation with FhGST induced a welldefined immune response, characterized by specific-FhGST antibody production and increased levels of circulating IL-4 after immunisation. These features were observed neither in the infected group nor in the control group. In addition, FhESP-specific IgG was observed in both immunised and non-immunised groups only after experimental challenge, suggesting that no cross-reactivity exists between FhGST and FhESP. The induction of specific-FhGST antibodies after immunisation and the role of those antibodies have been controversial. In a previous study a high antibody response following immunisation with GST was observed in sheep [11], whereas no anti-GST antibodies production was detected in another experiment with vaccinated and experimentally F. hepatica infected sheep [20]. A high production of anti-GST antibodies was observed in cattle, only when the animals were immunised with GST and some adjuvants, whereas no anti-GST antibodies were induced when other adjuvants were used. It was also observed that acquired resistance might be obtained in the absence of anti-GST antibodies, suggesting that these antibodies may not play a determinant role on protective responses [9]. Production of IFN-γ and IL-4 during the immunisation and the infection period of the experiment was also analyzed. We have developed a specific procedure to detect cytokines in plasma, which has not previously been used in ruminants during F. hepatica infection. However, Soliman et al. described the detection of cytokines using a similar method in human fasciolosis [21]. And this same procedure of studying plasmatic levels of IFN-γ and IL-4
has previously been reported by other authors in cattle [22], sheep [23] and goats [24,25]. To our knowledge, this is the first report on cytokine production in goat fasciolosis, and our most relevant findings are the elevation of plasmatic levels of IL-4 after immunisation with FhGST and the lack of significant modifications on plasmatic levels of both IL-4 and IFN-γ during the infection. In cattle, the kinetics of IL-4 and IFN-γ production during F. hepatica infection has been widely studied and it has been demonstrated that at the initial stages of the infection both IFN-γ and IL-4 are produced [26–28], but, as infection progresses, IFNγ production is suppressed [26], while IL-4 is produced throughout infection [29]. Recently, Flynn and Mulcahy have reported that IL-4 production is also reduced during the chronic stage of the infection [30]. Studies on cytokine kinetics in sheep are much more limited, but the obtained data are similar to those of cattle: in the first weeks of infection, a limited IFN-γ production has been observed [20,31], since in the chronic stage, IL4 gene expression is up regulated and IFN-γ down regulated [32]. IL-10 production has also been demonstrated in both bovine and ovine host throughout infection [29,31] and all these data on cytokine production, together with the evidence of eosinophilia [33] and a predominant IgG1 isotype production [19,26] indicates that immune response in bovine and ovine fasciolosis is a characteristic Th0/Th2 response. Further studies are needed to establish if immune response in caprine fasciolosis can also be considered as Th0/Th2 type. Some recent works have reported an important role for Tregulatory cytokines IL-10 and TGF-β in the development of the immune responses to F. hepatica infection in both cattle [30] and sheep [32]. Flynn and Mulcahy have demonstrated that both IL-10 and TGF-β have a role in controlling IL-4 and IFN-γ production during acute and chronic stage of the disease in cattle [30], suggesting that some regulatory mechanism may be limiting the host immune
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response and that some parasite molecules may be implicated in that regulation [29], as it has been demonstrated in the murine model [34]. In the sheep, Hacariz et al. related expression levels of IL-10 and TGF-β with susceptibility to infection, in terms of fluke burden. The different gene expression of these cytokines was not directly linked with differences in IL-4 and IFN-γ expression [32]. They suggested that down regulation of T-regulatory cytokines genes is a factor involved in the higher susceptibility to infection observed in some individuals. In our study, we have not determined the levels of IL-10 and TGF-β during the infection, and, in fact, there are very few studies related to these regulatory cytokines in goats [35], but their role in controlling IFN-γ and IL-4 during goat fasciolosis certainly deserves further investigations. Interestingly, in our experiment an elevation of IL-4 production 8 weeks after initial immunisation was found. Information about expression of IL-4 during vaccination trials in ruminants is scarce and contradictory. With other pathogen-host models have been described that immunisation with a protein-formulated vaccine was capable of inducing a significant production of IL-4 mRNA [36], however other studies have found no influence of vaccination on IL-4 mRNA production [37]. In conclusion, immunisation with native GST of F. hepatica in goats induces a clear humoral and cellular response, which did not result in a protective mechanism against the establishment of F. hepatica after infection. Since this is the first report of a vaccine trial using the GST antigen in caprine fasciolosis, more studies are required with recombinant GST products and different delivery formulations to complete the evaluation of this vaccine candidate in goats. Acknowledgements This work was supported by grant BIO-216 from the Andalusian Community Government, grant AGL2002-02777 from the Education and Science Ministry of Spain, and grant FOOD-CT-2005-023025DELIVER from the European Commission. References [1] Spithill TW, Smooker PM, Copeman DB. Fasciola gigantica: epidemiology, control, immunology and molecular biology. In: Dalton JP, editor. Fasciolosis. Wallingford: CAB International Publishing; 1999. p. 465–525. [2] Mas-Coma S. Epidemiology of fasciolosis in human endemic areas. J Helminthol 2005;79:207–16. [3] Martinez A, Martinez-Cruz S, Martinez FJ, Gutierrez PN, Hernandez S. Detection of antibodies of Fasciola hepatica excretory–secretory antigens in experimentally infected goats by enzyme immunosorbent assay. Vet Parasitol 1996;62:247–52. [4] Brennan GP, Fairwheather I, Trudgett A, Hoey E, McCoy, McConville M, et al. Understanding triclabendazole resistance. Exp Mol Pathol 2007;82:104–9. [5] Almeida MS, Torloni H, Lee-Ho P, Vilar MM, Thaumaturgo N, Simpson AJ, et al. Vaccination against Fasciola hepatica infection using a Schistosoma mansoni defined recombinant antigen, Sm14. Parasite Immunol 2003;3:135–7. [6] Muro A, Ramajo V, López J, Simón F, Hillyer GV. Fasciola hepatica: vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins. Vet Parasitol 1997;69:219–29. [7] Piacenza L, Acosta D, Basmadjian I, Dalton JP, Carmona C. Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep. Infect Immun 1999;67:1954–61. [8] Dalton JP, McGonigle S, Rolph TP, Andrews SJ. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin. Infect Immun 1996;64:5066–74. [9] Morrison CA, Thierry C, Sexton JL, Bowen F, Wicker J, Friedel T, et al. Protection of cattle against Fasciola hepatica infection by vaccination with glutathione Stransferase. Vaccine 1996;14:1603–12. [10] Zafra R, Buffoni L, Martínez-Moreno A, Péres-Écija A, Martínez-Moreno FJ, Pérez J. A study of the liver of goats immunised with a synthetic peptide of the Sm14 antigen and challenged with Fasciola hepatica. J Comp Pathol 2008;139:169–76. [11] Sexton JL, Milner AR, Panaccio M, Waddington J, Wijffels G, Chandler D, et al. Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep. J Immunol 1990;145:3905–10.
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