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Echinostoma caproni: Kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice
Experimental Parasitology 116 (2007) 390–398 www.elsevier.com/locate/yexpr
Echinostoma caproni: Kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice Javier Sotillo a, Carla Mun˜oz-Antoli a, Antonio Marcilla a, Bernard Fried b, J. Guillermo Esteban a, R. Toledo a,* a
Departamento de Parasitologı´a, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andre´s Estelle´s s/n, 46100 Burjassot-Valencia, Spain b Department of Biology, Lafayette College, Easton, PA 18042, USA Received 23 January 2007; received in revised form 12 February 2007; accepted 12 February 2007 Available online 28 February 2007
Abstract The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Echinostoma caproni; Mouse, Rat; IgM, immunoglobulin M; IgA, immunoglobulin A; IgG, immunoglobulin G; IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; IgG3, immunoglobulin G3; IFN-c, interferon-c; Interleukin-6, IL-6; Excretory–secretory, ES
1. Introduction Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host (Fried and Huffman, 1996). After infection, metacercariae excyst in the duodenum of the definitive host, and the juvenile worms migrate to the posterior third of the small intestine where they attach to the mucosa by the ventral sucker (Fried and Huffman, 1996; Fried and Graczyk, 2004). E. caproni has a wide range of definitive hosts, though the worm survival greatly differs between hosts. In ham*
Corresponding author. Fax: +34 963544769. E-mail address: [email protected] (R. Toledo).
0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.02.008
sters and mice, the infections are long-lasting, while in rats the infection is expelled at 7 8 week post-infection (wpi) (Odaibo et al., 1988, 1989; Christensen et al., 1990; Hansen et al., 1991; Mahler et al., 1995; Toledo et al., 2004a). The different compatibilities to E. caproni observed in different rodent species allows these host–parasite systems to be highly suitable for elucidating aspects of the host-specific components that determine the course of infections with intestinal trematodes (Toledo and Fried, 2005). The existing literature provides little information on the host- and parasite-related factors leading to the development of chronic E. caproni infections or, in contrast the rapid expulsion of the parasite. Earlier studies have shown that hosts in which chronic infections are developed are characterized by high inflammatory local response, high
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itivity of the infection was investigated weekly in each of the infected animals as described by Toledo et al. (2003a).
levels of seroantigens and elevated IgG levels in the serum. In contrast, the infection in rats is characterized by low levels of local inflammation, seroantigens and systemic IgG (Graczyk and Fried, 1994; Toledo et al., 2004b, 2005, 2006a,b). However, there are several aspects of the immunological response against E. caproni that remain to be studied in detail, i.e., the intestinal antibody responses, the kinetics of IgG subclasses and the potential effect of the antibodies on the course of the infection (Toledo et al., 2006b). In this sense, the only data available are those reported by Agger et al. (1993) and Brunet et al. (2000). Agger et al. (1993) analyzed the kinetics of IgM, IgA and IgG in the serum and intestinal wall of E. caproni-infected mice, during the first 70 days post-infection (dpi) and in the intestinal luminal content at 28 dpi. Elevated levels of IgM, IgA and IgG were detected in the serum and intestinal wall and only and increased level of IgA was detected in the intestinal luminal content. Regarding the IgG subclasses, Brunet et al. (2000) analyzed the profiles of IgG1 and IgG2a in the serum of mice experimentally infected with E. caproni during the first 23 dpi and a weak increasing of IgG2a was detected. The aim of the present study was to investigate the isotype specific immune responses both at the systemic and mucosal levels in two host species of E. caproni (rats and mice) in which parasite survival differs markedly. The results obtained may be of interest to gain further insight into the host–parasite relationships in intestinal trematode infections and into the factors determining worm rejection or the development of long-lasting infections.
Blood was collected weekly from each animal belonging to group A and their respective controls by cardiac puncture under anaesthesia. After clotting of the blood overnight at 4 °C, serum was separated from the clot by centrifugation. The serum samples were stored at –20 °C until use.
2. Materials and methods
2.4. Intestinal lavages
2.1. Parasite and experimental infections
The presence of antibodies in the intestine of infected rats and mice was investigated biweekly on the animals of group B and the intestinal secretions were collected using a slight modification of the method described by Ben-Smith et al. (1999). Briefly, at each time post-infection 3 mice and 3 rats were killed and the intestine removed. Three-millilitres of a solution of 0.1 mg/ml soybean trypsin–chymotrypsin inhibitor (Sigma) in 50 mM ethylenediaminetetraacetic acid (Sigma) was flushed through the intestine which was massaged gently before recovery of the fluid. The samples were then centrifuged at 700g for 10 min before addition of 30 ll of 100 mM PMSF in 95% ethanol. The samples were clarified by centrifugation at 3000 g at 4 °C for 15 min, after which 20 ml of PMSF and 20 ml of 1% sodium azide (Sigma) were added to the supernatant. The samples were stored at 20 °C until use.
The strain of E. caproni has been previously described by Hosier and Fried (1991). Encysted metacercariae of E. caproni were removed from the kidneys and pericardial cavities of experimentally infected Biomphalaria glabrata snails and used to infect mice (ICR) and albino rats (Wistar). Each of 28 male mice, weighing 32 40 g, and 28 rats, weighing 100 120 g, was infected by stomach tube with metacercariae of E. caproni. The infective doses were 75 and 100 metacercariae/animal for mice and rats, respectively. The animals were randomly allocated into groups A (10 mice and 10 rats) and B (18 mice and 18 rats). Group A was used to analyze the kinetics of serum antibodies against E. caproni during the first 12 wpi. Animals of group B were used to study the kinetics of antibodies in the intestine during the same time period. Moreover, 8 mice and 8 rats were left uninfected. From the uninfected animals, blood was collected weekly from 5 mice and 5 rats and used as controls for the analysis of the serum antibodies, and 3 mice and 3 rats were necropsied and used as controls for the study of intestinal antibodies. All the animals were maintained under conventional conditions with food and water ad libitum. The parasite egg release to determine pos-
2.2. Excretory/secretory (ES) antigens To obtain excretory/secretory antigens of E. caproni, we followed the methods of Toledo et al. (2003b). Briefly, adult worms were collected from the intestines of experimentally infected hamsters at 4 wpi. After thorough washing of the worms with phosphate buffered saline (PBS, pH 7.4), they were maintained in medium at concentrations of 10 worms/ml for 12 h at 37° C in PBS containing 0.8 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, Missouri), 100 U penicillin (Sigma), and 100 lg/ml streptomycin (Sigma). The medium was collected and centrifuged at 15,000g for 30 min at 4 °C and the supernatant was collected. The protein content was measured by the Bio-Rad (Hercules, California) protein assay and adjusted to 1 mg/ml using an ultrafiltration membrane (YM-3, Millipore, Bellerica, Massachusetts). The antigens were stored at –20 °C until use. 2.3. Serum samples
2.5. Indirect ELISA for antibody detection In order to detect specific antibodies against E. caproni ES products an indirect ELISA was carried out as described by Toledo et al. (2003b) with some modifications. This method was used to detect IgM, IgA, IgG; IgG1 and IgG2a in serum and intestinal samples of mice and rats and also IgG3 in
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serum samples from mice. Optimal dilutions of antibodies and sera were determined by checkerboard titrations. Polystyrene microtiter plates (Nalgene, Naperville, Illinois) were coated overnight at 4 °C with 100 ll/well of a 30 lg/ml solution of ES antigens of E. caproni in 0.1 M carbonate coating buffer, pH 9.6. The plates were washed three times with PBST. Uncoated sites were blocked with 5% non-fat dry milk in PBST. After incubation for 1 h at 37 °C, the plates were emptied and serum and intestinal samples at adequate dilution in PBST were dispensed into each well. The plates were incubated for 2 h at room temperature (RT) and then washed five times with PBST. One-hundred microliters of horse radish peroxidase-conjugated goat anti-mouse IgM (OEM concepts, Toms River, New Jersey) or anti-rat (OEM concepts), goat anti-mouse IgA (Nordic Immunological Laboratories, Tilburg, The Netherlands) or anti-rat (Nordic), goat anti-mouse IgG (Bio-Rad) or anti-rat (ICN Biomedicals, Aurora, Ohio), goat anti-mouse IgG1 (Nordic) or anti-rat (Nordic), goat anti-mouse IgG2a (Nordic) or anti-rat (Nordic) and goat anti-mouse IgG3 (Nordic) at adequate dilutions in PBS were added to each well and incubated for 2 h at RT. The plates were washed as described above and 100 ll of the substrate solution (10-ll hydrogen peroxide, 30% w/v + 25 ml of 0.1 M citrate buffer, pH 5.0 + 10 mg o-phenylenediamine hydrochloride) were added and incubated in the dark at RT. The enzyme reaction was stopped with 50 ll per well of 3 N HCl. The plates were read at 492 nm in a Bio-Rad 550 microplate ELISA reader. 2.6. Measurement of albumin The levels of serum and intestinal albumin were determined biweekly using by a modification of the method of Doumas et al. (1971) using the albumin reagent bromocresol green (Quanticromä albumin assay, BioAssays Systems, Hayward, California) according to the procedure sheet. Adequate blanks and controls were used accordingly. 2.7. Statistical analysis Each ELISA assay was performed in triplicate and the absorbance readings from wells with the same sample were expressed as the mean ± SD. The difference between the optical density (OD) values for the infection and control in serum specimens on each point in time was calculated and tested by the use of Student’s t-test. The same test was used to test the difference between the values in intestinal samples obtained in each 2-week period with respect to those in uninfected controls at time 0. P < 0.05 was considered as significant. 3. Results 3.1. Infection All mice and rats experimentally exposed to metacercariae of E. caproni were infected as determined by egg exam-
ination. The duration of the pre-patent period was uniform. Egg release began 9 12 (10.1 ± 0.4) dpi in mice and 10 12 (11.34 ± 0.44) dpi in rats. All the rats reverted had reverted to negative values at the 8 wpi. 3.2. Serum antibody responses Immunoglobulin M: The experimentally infected mice and rats developed significant IgM responses in serum against E. caproni (Fig. 1). In mice, the IgM levels rapidly increased to reach the maximum OD values at 2 wpi (0.317 ± 0.06) (Fig. 1a). Thereafter, the values progressively decreased and were similar to those of control mice at 11 wpi. Statistical significant differences (P < 0.05) with respect to control animals were observed for each sample analyzed from 2 to 9 wpi. The kinetics of IgM in rats were similar to those in mice (Fig. 1b). The IgM values rapidly increased to reach a maximum in the first week of the infection (0.384 ± 0.07). From this time post-infection, the values steadily declined and became similar to uninfected rats at 9 wpi. The OD values were significantly higher (P < 0.05) than those in control rats in the samples studied from 1 to 8 wpi. Immunoglobulin A: The levels of IgA in the serum of infected mice progressively increased over time to peak in the last week of the experiment (0.502 ± 0.08) (Fig. 1c). The OD values in the serum of infected mice were significantly higher (P < 0.05) than those in control mice for each week from 4 wpi until the end of the experiment. In the serum of infected rats, a sudden increase of the IgA levels was observed at 1 wpi, reaching the maximum value (0.191 ± 0.03) (Fig. 1d). Beyond this time, the values rapidly decreased and from 3 wpi they were similar to those of the control animals. Statistically significant differences with respect to controls were observed only at 1 and 2 wpi (P < 0.05). Immunoglobulin G subclasses: The kinetics observed in the study of serum IgG class and IgG subclasses in mice are shown in the Fig. 2. The results show that mice developed an intense response of total specific IgG against E. caproni (Fig. 2a). Antibodies were detected in all the infected mice from 2 wpi and the values progressively increased over the course of the experiment to reach a maximum value in the last week (1.142 ± 0.21). Statistically significant differences (P < 0.05) with respect to the respective controls were observed from 2 wpi until the end of the experiment. The kinetics of IgG1 in the serum of mice were similar to those of the total IgG (Fig. 2b). The OD values progressively increased during the course of the experiment. The maximum value was observed at 11 wpi (1.024 ± 0.11) and significant differences with uninfected mice were obtained for each sample collected from 3 to 12 wpi. In contrast, no IgG2a response was observed in the serum of infected mice (Fig. 2c). The values of infected mice were similar to those of controls during the entire experiment. The kinetics of IgG3 against E. caproni was studied in the serum of mice (Fig. 3d). A marked increase
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Fig. 1. Detection of serum IgM to Echinostoma caproni excretory/secretory antigens by indirect ELISA in experimentally infected mice (a) and rats (b) and serum IgA in experimentally infected mice (c) and rats (d). Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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Fig. 2. Detection of total IgG (a), IgG1 (b), IgG2a (c) and IgG3 (d) to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the serum of experimentally infected mice. Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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of IgG3 was observed from 2 wpi reaching a maximum at 8 wpi (0.689 ± 0.07). From 11 wpi and beyond the values declined. Statistically significant differences with respect to controls only were observed from 2 wpi until the end of the experiment (P < 0.05). The kinetics of total IgG and IgG subclasses in the serum of rats was different than in mice (Fig. 3). Infected rats only showed slow and weak total IgG responses against E. caproni (Fig. 3a). Weak increases were observed from 6 to 11 wpi. The OD values were negative at 12 wpi. The maximum value was observed at 8 wpi (0.376 ± 0.06) and significant differences (P < 0.05) with respect to control rats were observed in the sera collected from 7 to 11 wpi. The kinetics of IgG1 in rats were similar than those of total IgG (Fig. 3b). Slight increases were observed from 7 to 11 wpi and the maximum was detected at 8 wpi (0.245 ± 0.03). Statistically significant differences (P < 0.05) with respect to uninfected rats were detected in the samples collected from 7 to 11 wpi. The values of IgG2a in rats were slightly elevated during most of the entire experiment (Fig. 3c). The IgG2a levels rapidly increased and were uniform until 12 wpi. The maximum value was found at 11 wpi (0.178 ± 0.02) and statistically significant differences (P < 0.05) with respect to the respective controls were detected for all the samples colleted except those obtained at 3, 4, 7 and 9 wpi, probably in relation to the high degree of variability detected in the samples collected at these weeks.
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3.3. Intestinal antibody responses Immunoglobulin M: The kinetics of IgM in the intestinal fluid of rats and mice was rather different (Fig. 4a, b). In rats, no intestinal IgM reaction was observed during the experiment. In contrast, mice exhibited a late IgM response. The OD values suddenly increased at 8 wpi, reaching a maximum value of 0.278 ± 0.05. The values remained high until 12 wpi. Statistically significant differences (P < 0.05) with respect to uninfected mice were observed from 2 wpi until the end of the experiment. Immunoglobulin A: The kinetics of secretory IgA were also different between rats and mice (Fig. 4c, d). Rats did not exhibit an intestinal IgA response against E. caproni, whereas in mice the response was rapid and intense. The OD values in mice were significantly higher (P < 0.01) than those in the uninfected controls in all the samples analyzed from 2 wpi until the end of the experiment. The maximum value was observed at 8 wpi (1.232 ± 0.21). Immunoglobulin G subclasses: Mice exhibited an intense but slow total IgG response in the intestine (Fig. 5a). Increases in total IgG levels were observed from 6 wpi and beyond. Thereafter, the values remained high until the end of the experiment. The maximum value was detected at 12 wpi (0.781 ± 0.18). Statistically significant differences with respect to uninfected mice were observed from 6 wpi until the end of the experiment (P < 0.05).
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Weeks post-infection Fig. 3. Detection of total IgG (a), IgG1 (b) and IgG2a (c) to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the serum of experimentally infected rats. Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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Fig. 4. Detection of intestinal IgM to Echinostoma caproni excretory/secretory antigens by indirect ELISA in experimentally infected mice (a) and rats (b) and intestinal IgA in experimentally infected mice (c) and rats (d). Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
The curve of intestinal IgG1 in mice was similar to that of the total IgG (Fig. 5b). A sudden rise was observed at 8 wpi, reaching a peak (0.332 ± 0.07) and the values remained uniform until the end of the experiment. The OD values were significantly higher (P < 0.05) than in uninfected mice in the samples studied at 8, 10 and 12 wpi. Mice also exhibited a detectable intestinal IgG2a response against E. caproni (Fig. 5c). The OD values increased from 4 wpi reaching a maximum (0.160 ± 0.07). Thereafter, the values steadily declined to become negative from 10 and 12 wpi. Statistically significant differences (P < 0.01) with respect to the respective controls were detected for the samples colleted at 4, 6 and 8 wpi. Statistically significant higher (P < 0.05) values of total IgG in the intestine of infected rats with respect to uninfected rats were observed from 8 wpi until the end of the experiment (Fig. 5d). The maximum value was found at 12 wpi (0.140 ± 0.02). In contrast, no IgG1 response was detected (Fig. 5e). The values of IgG2a increased from 4 wpi to reach a maximum at 8 wpi (0.396 ± 0.05). Beyond 8 wpi, the values declined until the end of the experiment. Statistically significant differences with respect to uninfected rats were observed in the samples analyzed from 4 to 10 wpi (P < 0.05) (Fig. 5f).
3.4. Albumin determination To determine if the presence of immunoglobulins in the intestinal fluid was due to leakage into the gut from the serum or whether they were secreted locally, an assessment of albumin in serum and intestinal fluid was conducted in
both control and infected animals. The results show that there was no evidence of leakage from the serum into the gut. The levels of albumin in the intestinal samples were in all cases lower than the detection limit of the test (1 mg/ml). In contrast, the values of albumin in serum ranged from 29 52 mg/ml in rats and 19 34 mg/ml in mice.
4. Discussion To examine the potential role of antibody responses in host-protective immunity on primary E. caproni infections, we studied the serum and intestinal antibody kinetics in two host species exhibiting different patterns of E. caproni infection. Rats develop a marked capacity to expel primary infections of E. caproni and the worms are rapidly expelled (Hansen et al., 1991; Toledo et al., 2004a). In contrast, this species of echinostome evokes long-lasting infections in mice. In this context, the analysis of the antibody responses in both the systemic and local level in rats and mice may be of interest. Both host species showed specific antibody responses against E. caproni. Moreover, the absence of detectable levels of albumin in the intestinal fluids of infected rats and mice indicates that antibodies detected in these samples are produced locally. In general, mice developed earlier and more intense reactions than rats in both serum and intestinal samples. The early response in serum was of the IgM class in both host species, showing the maximum IgM values at 1 and 2 wpi in rats and mice, respectively. This agrees with the study of Agger et al. (1993) in which a significant increase of IgM levels were detected at 2 wpi in E. caproni-infected
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Fig. 5. Detection of total IgG, IgG1 and IgG2a to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the intestine of experimentally infected mice (a, b and c, respectively) and rats (d, e and f, respectively). Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
mice. The rapid and intense IgM response, together with the low levels IgG detected in rats, suggests the presence of thymus-independent antigens in E. caproni adult worms. This is supported by the intense IgG3 response observed in mice. Responses mediated by IgG3 are related to carbohydrate epitopes (Snapper et al., 1992; Dea-Ayuela et al., 2000). It has been shown that certain thymus-independent antigens induce the CD5+ populations to undergo a IgG3 class switch (Drabek et al., 1997) upon control of Th1 cytokine IFN-c (Snapper et al., 1992). The intense IgG3 response observed in mice could be related to carbohydrate antigens released from adult worms and adsorbed throughout the intestinal mucosa (Andresen et al., 1989; Toledo et al., 2005, 2006a). Although IgM can be produced locally, intestinal responses were slower and less intense than those in serum. Agger et al. (1993) did not detect IgM in the intestinal content of E. caproni-infected mice at 4 wpi. The late rise of the intestinal IgM observed in the present study may be an explanation of the negative results obtained by Agger
et al. (1993). This late rise in local IgM raises the possibility that this response may be involved in the reduced worm burden observed in E. caproni-infected mice from 8 to 10 wpi (Odaibo et al., 1988). The IgM class is a major complement fixing antibody and complement has been suggested as one of the potential effector immune mechanisms against E. caproni (Simonsen and Andersen, 1986). Although this possibility cannot be disregarded completely, the lack of intestinal IgM response in rats and the earlier worm rejection in this host suggest that the role of local IgM response in parasite clearance could be considered only as secondary and other effector mechanisms should be implicated. The kinetics of total specific IgG observed in the serum of rats and mice are consistent with earlier studies (Agger et al., 1993; Graczyk and Fried, 1994; Toledo et al., 2004b,2005). The serum IgG response was markedly greater in mice than in rats probably in relation to a greater intestinal antigen uptake in relation to a higher local inflammation (Toledo et al., 2006a). Moreover, the kinetics of serum IgG1 and IgG2a were also different
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between both host species. Our results suggest a markedly biased response toward a Th2 phenotype and characterized by a strong production of IgG1 from 3 wpi and beyond. In rats an initial dominance of IgG2a response was observed. However, slight increase of IgG1 was detected from 7 wpi suggesting that early expulsion of E. caproni might be associated with balanced Th1/Th2 systemic responses. Brunet et al. (2000) analysed the IgG1 and IgG2a profiles in the serum of E. caproniinfected mice during the first 23 dpi and found a weak dominance of IgG2a on the last day of their experiment. These authors suggested that the development of chronic E. caproni infections was associated with Th1 responses. The discrepancies with respect to our results could be related to different methods used in each study. Brunet et al. (2000) only studied the initial phases of the infection. Our results show that during these phases the levels of IgG1 and IgG2a are low and no marked differences between both subclasses are established. However, at 3 wpi and beyond a progressive increase of IgG1 levels occurs. In fact, Brunet et al. (2000) did not detect changes in CD4+ and CD8+ cell populations and similar increases in both Th1 (IFN-c) and Th2 (IL-4) cytokines were observed during this initial period of the infection. In contrast to earlier studies, our results indicate that the development of long-lasting E. caproni infections is associated with dominance of systemic Th2 responses with high levels of serum IgG1 subclass. The IgG1 and IgG2a intestinal responses in mice and rats were slower and of lesser intensity than in serum. In mice, increases in both IgG1 and IgG2a subclasses were observed which could reflect a balanced Th1/Th2 local response. In contrast, rats only developed IgG2a response which suggests that local cellular responses could be of great importance in the parasite expulsion as previously suggested (Toledo et al., 2006a). Interestingly, the worm expulsion coincides with the pike of IgG2a observed in rats at 7 8 wpi. Although little is known about the mechanisms operating in natural hosts of E. caproni, cellular mechanisms could be of great importance in determining the course of the infection. Circulating and secretory IgA responses have been related to expulsion of intestinal protozoan and helminth parasite (Ben-Smith et al., 1999; Langford et al., 2002; Miquel et al., 2005). However, our results suggest that increases of IgA are not sufficient for E. caproni rejection. The infection in mice is able to stimulate high and rapid IgA antibody responses at both systemic and local levels although the survival of E. caproni is higher than in rats that do not exhibit IgA response. The induction of IgA response in mice might be the consequence of IL-6 production in response to exposure to E. caproni antigens, a Th2 cytokine that has been implicated in the switching to IgA production (Mazanec et al., 1993; Goodrich and McGee, 1999). In this context, the dominance of the Th2 responses observed in the serum of mice could explain the high IgA reactivity against E. caproni.
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Echinostoma caproni adults are attached to the host intestinal mucosa. Therefore, effective host defense against this digenean requires effector mechanisms that are active at the host mucosa. Our study suggests that the development of long-lasting E. caproni infection is related with dominance of Th2 systemic responses that appear not to be effective against the parasite. However, early expulsion of these worms from rodent hosts could be related to the generation of local Th1 responses involving cellular mechanisms. Acknowledgements The study was supported by the projects CGL200502321/BOS from the Ministerio de Educacio´n y Ciencia (Spain), GV04B/107 and GV05/039 from the Conselleria d’Empresa, Universitat I Cie`ncia de la Generalitat Valenciana (Spain) and the Project UV-AE-20050201 de la Universitat de Vale`ncia (Spain). This work has been carried out while the first author (J.S.) was recipient of a pre-doctoral fellowship from the Ministerio de Educacio´n y Ciencia, Madrid (Spain). This research complies with the current laws for animal health research in Spain. References Agger, M.K., Simonsen, P.E., Vennervald, B.J., 1993. The antibody response in serum, intestinal wall and intestinal lumen of NMRI mice infected with Echinostoma caproni. Journal of Helminthology 67, 169–178. Andresen, K., Simonsen, P.E., Andersen, B.J., Birch-Andersen, A., 1989. Echinostoma caproni in mice, shedding of antigens from the surface of an intestinal trematode. International Journal for Parasitology 19, 111–118. Ben-Smith, A., Wahid, F.N., Lammas, D.A., Behnke, J.M., 1999. The relationships between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection. Parasite Immunology 21, 383–395. Brunet, L.R., Joseph, S., Dunne, D.W., Fried, B., 2000. Immune responses during the acute stages of infection with the intestinal trematode Echinostoma caproni. Parasitology 120, 565–571. Christensen, N.Ø., Simonsen, P., Odaibo, A.B., Mahler, H., 1990. Establishment, survival and fecundity in Echinostoma caproni (Trematoda) infections in hamsters and jirds. Journal of the Helminthological Society of Washington 57, 104–107. Dea-Ayuela, M.A., Martı´nez-Ferna´ndez, A.R., Bola´s-Ferna´ndez, F., 2000. Comparison of IgG3 responses to carbohydrates following mouse immunization with six species of Trichinella. Journal of Helminthology 74, 215–223. Doumas, B., Watson, W., Biggs, H., 1971. Albumin standards and the measurement of serum albumin with bromocresol green. Clinica Chimica Acta 31, 87–96. Drabek, D., Raguz, S., De Wit, T.P., Dingjan, G.M., Savelkoul, H.F., Grosveld, F., Hendriks, R.W., 1997. Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under the control of the class II major histocompatibility complex Ea locus control region. Proceedings of the National Academy of Sciences 94, 610–615. Fried, B., Graczyk, T.K., 2004. Recent advances in the biology of Echinostoma species in the ‘‘revolutum’’ group. Advances in Parasitology 58, 139–195. Fried, B., Huffman, J.E., 1996. The biology of the intestinal trematode Echinostoma caproni. Advances in Parasitology 38, 311–368.
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Goodrich, M.E., McGee, D.W., 1999. Effect of intestinal epithelial cell cytokines on mucosal B-cell IgA secretion: enhancing effect of epithelial-derived IL-6 but not TGF-beta on IgA+B cells. Immunology Letters 67, 11–14. Graczyk, T.K., Fried, B., 1994. ELISA method for detecting antiEchinostoma caproni (Trematoda) antibodies in experimentally infected ICR mice. Journal of Parasitology 80, 544–549. Hansen, K., Nielsen, J.W., Hindsbo, O., Christensen, N.Ø., 1991. Echinostoma caproni in rats: worm population dynamics and host blood eosinophilia during primary 6, 25 and 50 metacercarial infections, and resistance to secondary and superimposed infections. Parasitology Research 77, 686–690. Hosier, D.W., Fried, B., 1991. Infectivity, growth and distribution of Echinostoma revolutum (Trematoda) in the ICR mice. Journal of Parasitology 77, 640–642. Langford, T.D., Housley, M.P., Boes, M., Chen, J., Kagnoff, M.F., Gillin, F.D., Eckmann, L., 2002. Central importance of immunoglobulin A in host defense against Giardia spp.. Infection and Immunity 70, 11–18. Mahler, H., Christensen, N.Ø., Hindsbo, O., 1995. Studies on the reproductive capacity of Echinostoma caproni (Trematoda) in hamsters and jirds. International Journal for Parasitology 25, 705–710. Mazanec, M.B., Nedrud, J.G., Kaetzel, C.S., Lamm, M.E., 1993. A threetiered view of the role of IgA in mucosal defense. Immunology Today 14, 430–435. Miquel, N., Roepstorff, A., Bailey, M., Eriksen, L., 2005. Host immune reactions and worm kinetics during the expulsion of Ascaris suum in pigs. Parasite Immunology 27, 79–88. Odaibo, A.B., Christensen, N.Ø., Ukoli, F.M.A., 1988. Establishment survival and fecundity in Echinostoma caproni (Trematoda) infections in NMRI mice. Proceedings of the Helminthological Society of Washington 55, 265–269. Odaibo, A.B., Christensen, N.Ø., Ukoli, F.M.A., 1989. Further studies on the population regulation in Echinostoma caproni infections in NMRI mice. Proceedings of the Helminthological Society of Washington 56, 192–198. Simonsen, P.E., Andersen, B.J., 1986. Echinostoma revolutum in mice; dynamics of the antibody attack to the surface of an intestinal trematode. International Journal for Parasitology 16, 475–482.
Snapper, C.M., McIntyre, T.M., Mandler, R., Pecanha, L.M.T., Finkelman, F.D., Mond, J.J., 1992. Induction of IgG3 secretion by interferon-c: a model for T cell-independent class switching in response to T cell-independent Type 2 antigens. Journal of Experimental Medicine 175, 1367–1371. Toledo, R., Fried, B., 2005. Echinostomes as experimental models in adult parasite-vertebrate host interactions. Trends in Parasitology 21, 251–254. Toledo, R., Espert, A., Carpena, I., Mun˜oz-Antoli, C., Esteban, J.G., 2003a. An experimental study of the reproductive success of Echinostoma friedi (Trematoda: Echinostomatidae) in the golden hamster. Parasitology 126, 433–441. Toledo, R., Espert, A., Mun˜oz-Antoli, C., Marcilla, A., Fried, B., Esteban, J.G., 2003b. Development of an antibody-based capture enzyme-linked immunosorbent assay for detecting Echinostoma caproni (Trematoda) in experimentally infected rats: kinetics of coproantigen excretion. Journal of Parasitology 89, 1227–1231. Toledo, R., Espert, A., Carpena, I., Mun˜oz-Antoli, C., Fried, B., Esteban, J.G., 2004a. The comparative development of Echinostoma caproni (Trematoda: Echinostomatidae) adults in experimentally infected hamsters and rats. Parasitology Research 93, 439–444. Toledo, R., Espert, A., Mun˜oz-Antoli, C., Marcilla, A., Fried, B., Esteban, J.G., 2004b. Kinetics of Echinostoma caproni (Trematoda: Echinostomatidae) antigens in feces and serum of experimentally infected hamsters and rats. Journal of Parasitology 90, 752–758. Toledo, R., Espert, A., Mun˜oz-Antoli, C., Marcilla, A., Fried, B., Esteban, J.G., 2005. Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae). Journal of Parasitology 91, 978–980. Toledo, R., Monteagudo, C., Espert, A., Fried, B., Esteban, J.G., Marcilla, A., 2006a. Echinostoma caproni: Intestinal pathology in the golden hamster, a highly compatible host, and the Wistar rat, a less compatible host. Experimental Parasitology 112, 164–171. Toledo, R., Esteban, J.G., Fried, B., 2006b. Immunology and pathology of intestinal trematodes in their definitive hosts. Advances in Parasitology 63, 285–365.
Echinostoma caproni: Kinetics of IgM, IgA and IgG subclasses in the serum and intestine of experimentally infected rats and mice Javier Sotillo a, Carla Mun˜oz-Antoli a, Antonio Marcilla a, Bernard Fried b, J. Guillermo Esteban a, R. Toledo a,* a
Departamento de Parasitologı´a, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andre´s Estelle´s s/n, 46100 Burjassot-Valencia, Spain b Department of Biology, Lafayette College, Easton, PA 18042, USA Received 23 January 2007; received in revised form 12 February 2007; accepted 12 February 2007 Available online 28 February 2007
Abstract The kinetics of specific immunoglobulin M, A and IgG subclasses against Echinostoma caproni (Trematoda: Echinostomatidae) were analyzed in serum and intestinal fluid of two host species (Wistar rats and ICR mice) in which the course of the infection markedly differs. In rats, the worms were rapidly expelled, whereas E. caproni evokes in mice long-lasting infection. The pattern of antibody responses in both serum and intestinal samples was different in each host species. Serum responses in mice were characterized by significant increases of IgM, IgA, total IgG, IgG1 and IgG3, but not IgG2a. In contrast, serum responses in rats showed elevated levels of IgM, probably in relation to thymus-independent antigens, and slight increases of total IgG, IgG1 and IgG2a. At the intestinal level, increases of IgM and IgA levels were observed in mice. In regard to IgG subclasses, increases in both IgG1 and IgG2a were detected. Later decreases to normal values in IgG2a were also detected. In rats, only increases in total IgG and IgG2a were found. According to our results the development of long-lasting E. caproni infections in mice appears to be associated with a dominance of Th2 responses at the systemic level and balanced Th1/Th2 responses at the local level, characterized by initial increases in IgG1 and IgG2a levels. In contrast, the worm expulsion appears to be related to increases in local IgG2a levels. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Echinostoma caproni; Mouse, Rat; IgM, immunoglobulin M; IgA, immunoglobulin A; IgG, immunoglobulin G; IgG1, immunoglobulin G1; IgG2a, immunoglobulin G2a; IgG3, immunoglobulin G3; IFN-c, interferon-c; Interleukin-6, IL-6; Excretory–secretory, ES
1. Introduction Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode with no tissue phases in the definitive host (Fried and Huffman, 1996). After infection, metacercariae excyst in the duodenum of the definitive host, and the juvenile worms migrate to the posterior third of the small intestine where they attach to the mucosa by the ventral sucker (Fried and Huffman, 1996; Fried and Graczyk, 2004). E. caproni has a wide range of definitive hosts, though the worm survival greatly differs between hosts. In ham*
Corresponding author. Fax: +34 963544769. E-mail address: [email protected] (R. Toledo).
0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.02.008
sters and mice, the infections are long-lasting, while in rats the infection is expelled at 7 8 week post-infection (wpi) (Odaibo et al., 1988, 1989; Christensen et al., 1990; Hansen et al., 1991; Mahler et al., 1995; Toledo et al., 2004a). The different compatibilities to E. caproni observed in different rodent species allows these host–parasite systems to be highly suitable for elucidating aspects of the host-specific components that determine the course of infections with intestinal trematodes (Toledo and Fried, 2005). The existing literature provides little information on the host- and parasite-related factors leading to the development of chronic E. caproni infections or, in contrast the rapid expulsion of the parasite. Earlier studies have shown that hosts in which chronic infections are developed are characterized by high inflammatory local response, high
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itivity of the infection was investigated weekly in each of the infected animals as described by Toledo et al. (2003a).
levels of seroantigens and elevated IgG levels in the serum. In contrast, the infection in rats is characterized by low levels of local inflammation, seroantigens and systemic IgG (Graczyk and Fried, 1994; Toledo et al., 2004b, 2005, 2006a,b). However, there are several aspects of the immunological response against E. caproni that remain to be studied in detail, i.e., the intestinal antibody responses, the kinetics of IgG subclasses and the potential effect of the antibodies on the course of the infection (Toledo et al., 2006b). In this sense, the only data available are those reported by Agger et al. (1993) and Brunet et al. (2000). Agger et al. (1993) analyzed the kinetics of IgM, IgA and IgG in the serum and intestinal wall of E. caproni-infected mice, during the first 70 days post-infection (dpi) and in the intestinal luminal content at 28 dpi. Elevated levels of IgM, IgA and IgG were detected in the serum and intestinal wall and only and increased level of IgA was detected in the intestinal luminal content. Regarding the IgG subclasses, Brunet et al. (2000) analyzed the profiles of IgG1 and IgG2a in the serum of mice experimentally infected with E. caproni during the first 23 dpi and a weak increasing of IgG2a was detected. The aim of the present study was to investigate the isotype specific immune responses both at the systemic and mucosal levels in two host species of E. caproni (rats and mice) in which parasite survival differs markedly. The results obtained may be of interest to gain further insight into the host–parasite relationships in intestinal trematode infections and into the factors determining worm rejection or the development of long-lasting infections.
Blood was collected weekly from each animal belonging to group A and their respective controls by cardiac puncture under anaesthesia. After clotting of the blood overnight at 4 °C, serum was separated from the clot by centrifugation. The serum samples were stored at –20 °C until use.
2. Materials and methods
2.4. Intestinal lavages
2.1. Parasite and experimental infections
The presence of antibodies in the intestine of infected rats and mice was investigated biweekly on the animals of group B and the intestinal secretions were collected using a slight modification of the method described by Ben-Smith et al. (1999). Briefly, at each time post-infection 3 mice and 3 rats were killed and the intestine removed. Three-millilitres of a solution of 0.1 mg/ml soybean trypsin–chymotrypsin inhibitor (Sigma) in 50 mM ethylenediaminetetraacetic acid (Sigma) was flushed through the intestine which was massaged gently before recovery of the fluid. The samples were then centrifuged at 700g for 10 min before addition of 30 ll of 100 mM PMSF in 95% ethanol. The samples were clarified by centrifugation at 3000 g at 4 °C for 15 min, after which 20 ml of PMSF and 20 ml of 1% sodium azide (Sigma) were added to the supernatant. The samples were stored at 20 °C until use.
The strain of E. caproni has been previously described by Hosier and Fried (1991). Encysted metacercariae of E. caproni were removed from the kidneys and pericardial cavities of experimentally infected Biomphalaria glabrata snails and used to infect mice (ICR) and albino rats (Wistar). Each of 28 male mice, weighing 32 40 g, and 28 rats, weighing 100 120 g, was infected by stomach tube with metacercariae of E. caproni. The infective doses were 75 and 100 metacercariae/animal for mice and rats, respectively. The animals were randomly allocated into groups A (10 mice and 10 rats) and B (18 mice and 18 rats). Group A was used to analyze the kinetics of serum antibodies against E. caproni during the first 12 wpi. Animals of group B were used to study the kinetics of antibodies in the intestine during the same time period. Moreover, 8 mice and 8 rats were left uninfected. From the uninfected animals, blood was collected weekly from 5 mice and 5 rats and used as controls for the analysis of the serum antibodies, and 3 mice and 3 rats were necropsied and used as controls for the study of intestinal antibodies. All the animals were maintained under conventional conditions with food and water ad libitum. The parasite egg release to determine pos-
2.2. Excretory/secretory (ES) antigens To obtain excretory/secretory antigens of E. caproni, we followed the methods of Toledo et al. (2003b). Briefly, adult worms were collected from the intestines of experimentally infected hamsters at 4 wpi. After thorough washing of the worms with phosphate buffered saline (PBS, pH 7.4), they were maintained in medium at concentrations of 10 worms/ml for 12 h at 37° C in PBS containing 0.8 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, Missouri), 100 U penicillin (Sigma), and 100 lg/ml streptomycin (Sigma). The medium was collected and centrifuged at 15,000g for 30 min at 4 °C and the supernatant was collected. The protein content was measured by the Bio-Rad (Hercules, California) protein assay and adjusted to 1 mg/ml using an ultrafiltration membrane (YM-3, Millipore, Bellerica, Massachusetts). The antigens were stored at –20 °C until use. 2.3. Serum samples
2.5. Indirect ELISA for antibody detection In order to detect specific antibodies against E. caproni ES products an indirect ELISA was carried out as described by Toledo et al. (2003b) with some modifications. This method was used to detect IgM, IgA, IgG; IgG1 and IgG2a in serum and intestinal samples of mice and rats and also IgG3 in
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serum samples from mice. Optimal dilutions of antibodies and sera were determined by checkerboard titrations. Polystyrene microtiter plates (Nalgene, Naperville, Illinois) were coated overnight at 4 °C with 100 ll/well of a 30 lg/ml solution of ES antigens of E. caproni in 0.1 M carbonate coating buffer, pH 9.6. The plates were washed three times with PBST. Uncoated sites were blocked with 5% non-fat dry milk in PBST. After incubation for 1 h at 37 °C, the plates were emptied and serum and intestinal samples at adequate dilution in PBST were dispensed into each well. The plates were incubated for 2 h at room temperature (RT) and then washed five times with PBST. One-hundred microliters of horse radish peroxidase-conjugated goat anti-mouse IgM (OEM concepts, Toms River, New Jersey) or anti-rat (OEM concepts), goat anti-mouse IgA (Nordic Immunological Laboratories, Tilburg, The Netherlands) or anti-rat (Nordic), goat anti-mouse IgG (Bio-Rad) or anti-rat (ICN Biomedicals, Aurora, Ohio), goat anti-mouse IgG1 (Nordic) or anti-rat (Nordic), goat anti-mouse IgG2a (Nordic) or anti-rat (Nordic) and goat anti-mouse IgG3 (Nordic) at adequate dilutions in PBS were added to each well and incubated for 2 h at RT. The plates were washed as described above and 100 ll of the substrate solution (10-ll hydrogen peroxide, 30% w/v + 25 ml of 0.1 M citrate buffer, pH 5.0 + 10 mg o-phenylenediamine hydrochloride) were added and incubated in the dark at RT. The enzyme reaction was stopped with 50 ll per well of 3 N HCl. The plates were read at 492 nm in a Bio-Rad 550 microplate ELISA reader. 2.6. Measurement of albumin The levels of serum and intestinal albumin were determined biweekly using by a modification of the method of Doumas et al. (1971) using the albumin reagent bromocresol green (Quanticromä albumin assay, BioAssays Systems, Hayward, California) according to the procedure sheet. Adequate blanks and controls were used accordingly. 2.7. Statistical analysis Each ELISA assay was performed in triplicate and the absorbance readings from wells with the same sample were expressed as the mean ± SD. The difference between the optical density (OD) values for the infection and control in serum specimens on each point in time was calculated and tested by the use of Student’s t-test. The same test was used to test the difference between the values in intestinal samples obtained in each 2-week period with respect to those in uninfected controls at time 0. P < 0.05 was considered as significant. 3. Results 3.1. Infection All mice and rats experimentally exposed to metacercariae of E. caproni were infected as determined by egg exam-
ination. The duration of the pre-patent period was uniform. Egg release began 9 12 (10.1 ± 0.4) dpi in mice and 10 12 (11.34 ± 0.44) dpi in rats. All the rats reverted had reverted to negative values at the 8 wpi. 3.2. Serum antibody responses Immunoglobulin M: The experimentally infected mice and rats developed significant IgM responses in serum against E. caproni (Fig. 1). In mice, the IgM levels rapidly increased to reach the maximum OD values at 2 wpi (0.317 ± 0.06) (Fig. 1a). Thereafter, the values progressively decreased and were similar to those of control mice at 11 wpi. Statistical significant differences (P < 0.05) with respect to control animals were observed for each sample analyzed from 2 to 9 wpi. The kinetics of IgM in rats were similar to those in mice (Fig. 1b). The IgM values rapidly increased to reach a maximum in the first week of the infection (0.384 ± 0.07). From this time post-infection, the values steadily declined and became similar to uninfected rats at 9 wpi. The OD values were significantly higher (P < 0.05) than those in control rats in the samples studied from 1 to 8 wpi. Immunoglobulin A: The levels of IgA in the serum of infected mice progressively increased over time to peak in the last week of the experiment (0.502 ± 0.08) (Fig. 1c). The OD values in the serum of infected mice were significantly higher (P < 0.05) than those in control mice for each week from 4 wpi until the end of the experiment. In the serum of infected rats, a sudden increase of the IgA levels was observed at 1 wpi, reaching the maximum value (0.191 ± 0.03) (Fig. 1d). Beyond this time, the values rapidly decreased and from 3 wpi they were similar to those of the control animals. Statistically significant differences with respect to controls were observed only at 1 and 2 wpi (P < 0.05). Immunoglobulin G subclasses: The kinetics observed in the study of serum IgG class and IgG subclasses in mice are shown in the Fig. 2. The results show that mice developed an intense response of total specific IgG against E. caproni (Fig. 2a). Antibodies were detected in all the infected mice from 2 wpi and the values progressively increased over the course of the experiment to reach a maximum value in the last week (1.142 ± 0.21). Statistically significant differences (P < 0.05) with respect to the respective controls were observed from 2 wpi until the end of the experiment. The kinetics of IgG1 in the serum of mice were similar to those of the total IgG (Fig. 2b). The OD values progressively increased during the course of the experiment. The maximum value was observed at 11 wpi (1.024 ± 0.11) and significant differences with uninfected mice were obtained for each sample collected from 3 to 12 wpi. In contrast, no IgG2a response was observed in the serum of infected mice (Fig. 2c). The values of infected mice were similar to those of controls during the entire experiment. The kinetics of IgG3 against E. caproni was studied in the serum of mice (Fig. 3d). A marked increase
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Fig. 1. Detection of serum IgM to Echinostoma caproni excretory/secretory antigens by indirect ELISA in experimentally infected mice (a) and rats (b) and serum IgA in experimentally infected mice (c) and rats (d). Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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Fig. 2. Detection of total IgG (a), IgG1 (b), IgG2a (c) and IgG3 (d) to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the serum of experimentally infected mice. Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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of IgG3 was observed from 2 wpi reaching a maximum at 8 wpi (0.689 ± 0.07). From 11 wpi and beyond the values declined. Statistically significant differences with respect to controls only were observed from 2 wpi until the end of the experiment (P < 0.05). The kinetics of total IgG and IgG subclasses in the serum of rats was different than in mice (Fig. 3). Infected rats only showed slow and weak total IgG responses against E. caproni (Fig. 3a). Weak increases were observed from 6 to 11 wpi. The OD values were negative at 12 wpi. The maximum value was observed at 8 wpi (0.376 ± 0.06) and significant differences (P < 0.05) with respect to control rats were observed in the sera collected from 7 to 11 wpi. The kinetics of IgG1 in rats were similar than those of total IgG (Fig. 3b). Slight increases were observed from 7 to 11 wpi and the maximum was detected at 8 wpi (0.245 ± 0.03). Statistically significant differences (P < 0.05) with respect to uninfected rats were detected in the samples collected from 7 to 11 wpi. The values of IgG2a in rats were slightly elevated during most of the entire experiment (Fig. 3c). The IgG2a levels rapidly increased and were uniform until 12 wpi. The maximum value was found at 11 wpi (0.178 ± 0.02) and statistically significant differences (P < 0.05) with respect to the respective controls were detected for all the samples colleted except those obtained at 3, 4, 7 and 9 wpi, probably in relation to the high degree of variability detected in the samples collected at these weeks.
a Optical density (490 nm)
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3.3. Intestinal antibody responses Immunoglobulin M: The kinetics of IgM in the intestinal fluid of rats and mice was rather different (Fig. 4a, b). In rats, no intestinal IgM reaction was observed during the experiment. In contrast, mice exhibited a late IgM response. The OD values suddenly increased at 8 wpi, reaching a maximum value of 0.278 ± 0.05. The values remained high until 12 wpi. Statistically significant differences (P < 0.05) with respect to uninfected mice were observed from 2 wpi until the end of the experiment. Immunoglobulin A: The kinetics of secretory IgA were also different between rats and mice (Fig. 4c, d). Rats did not exhibit an intestinal IgA response against E. caproni, whereas in mice the response was rapid and intense. The OD values in mice were significantly higher (P < 0.01) than those in the uninfected controls in all the samples analyzed from 2 wpi until the end of the experiment. The maximum value was observed at 8 wpi (1.232 ± 0.21). Immunoglobulin G subclasses: Mice exhibited an intense but slow total IgG response in the intestine (Fig. 5a). Increases in total IgG levels were observed from 6 wpi and beyond. Thereafter, the values remained high until the end of the experiment. The maximum value was detected at 12 wpi (0.781 ± 0.18). Statistically significant differences with respect to uninfected mice were observed from 6 wpi until the end of the experiment (P < 0.05).
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Weeks post-infection Fig. 3. Detection of total IgG (a), IgG1 (b) and IgG2a (c) to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the serum of experimentally infected rats. Mean optical density values (OD) of control (s) and infected (d) animals over the course of the experiment. Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
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Fig. 4. Detection of intestinal IgM to Echinostoma caproni excretory/secretory antigens by indirect ELISA in experimentally infected mice (a) and rats (b) and intestinal IgA in experimentally infected mice (c) and rats (d). Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
The curve of intestinal IgG1 in mice was similar to that of the total IgG (Fig. 5b). A sudden rise was observed at 8 wpi, reaching a peak (0.332 ± 0.07) and the values remained uniform until the end of the experiment. The OD values were significantly higher (P < 0.05) than in uninfected mice in the samples studied at 8, 10 and 12 wpi. Mice also exhibited a detectable intestinal IgG2a response against E. caproni (Fig. 5c). The OD values increased from 4 wpi reaching a maximum (0.160 ± 0.07). Thereafter, the values steadily declined to become negative from 10 and 12 wpi. Statistically significant differences (P < 0.01) with respect to the respective controls were detected for the samples colleted at 4, 6 and 8 wpi. Statistically significant higher (P < 0.05) values of total IgG in the intestine of infected rats with respect to uninfected rats were observed from 8 wpi until the end of the experiment (Fig. 5d). The maximum value was found at 12 wpi (0.140 ± 0.02). In contrast, no IgG1 response was detected (Fig. 5e). The values of IgG2a increased from 4 wpi to reach a maximum at 8 wpi (0.396 ± 0.05). Beyond 8 wpi, the values declined until the end of the experiment. Statistically significant differences with respect to uninfected rats were observed in the samples analyzed from 4 to 10 wpi (P < 0.05) (Fig. 5f).
3.4. Albumin determination To determine if the presence of immunoglobulins in the intestinal fluid was due to leakage into the gut from the serum or whether they were secreted locally, an assessment of albumin in serum and intestinal fluid was conducted in
both control and infected animals. The results show that there was no evidence of leakage from the serum into the gut. The levels of albumin in the intestinal samples were in all cases lower than the detection limit of the test (1 mg/ml). In contrast, the values of albumin in serum ranged from 29 52 mg/ml in rats and 19 34 mg/ml in mice.
4. Discussion To examine the potential role of antibody responses in host-protective immunity on primary E. caproni infections, we studied the serum and intestinal antibody kinetics in two host species exhibiting different patterns of E. caproni infection. Rats develop a marked capacity to expel primary infections of E. caproni and the worms are rapidly expelled (Hansen et al., 1991; Toledo et al., 2004a). In contrast, this species of echinostome evokes long-lasting infections in mice. In this context, the analysis of the antibody responses in both the systemic and local level in rats and mice may be of interest. Both host species showed specific antibody responses against E. caproni. Moreover, the absence of detectable levels of albumin in the intestinal fluids of infected rats and mice indicates that antibodies detected in these samples are produced locally. In general, mice developed earlier and more intense reactions than rats in both serum and intestinal samples. The early response in serum was of the IgM class in both host species, showing the maximum IgM values at 1 and 2 wpi in rats and mice, respectively. This agrees with the study of Agger et al. (1993) in which a significant increase of IgM levels were detected at 2 wpi in E. caproni-infected
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Fig. 5. Detection of total IgG, IgG1 and IgG2a to Echinostoma caproni excretory/secretory antigens by indirect ELISA in the intestine of experimentally infected mice (a, b and c, respectively) and rats (d, e and f, respectively). Vertical bars represent the standard deviation. Asterisks represent significant differences with respect to uninfected control animals (P < 0.05).
mice. The rapid and intense IgM response, together with the low levels IgG detected in rats, suggests the presence of thymus-independent antigens in E. caproni adult worms. This is supported by the intense IgG3 response observed in mice. Responses mediated by IgG3 are related to carbohydrate epitopes (Snapper et al., 1992; Dea-Ayuela et al., 2000). It has been shown that certain thymus-independent antigens induce the CD5+ populations to undergo a IgG3 class switch (Drabek et al., 1997) upon control of Th1 cytokine IFN-c (Snapper et al., 1992). The intense IgG3 response observed in mice could be related to carbohydrate antigens released from adult worms and adsorbed throughout the intestinal mucosa (Andresen et al., 1989; Toledo et al., 2005, 2006a). Although IgM can be produced locally, intestinal responses were slower and less intense than those in serum. Agger et al. (1993) did not detect IgM in the intestinal content of E. caproni-infected mice at 4 wpi. The late rise of the intestinal IgM observed in the present study may be an explanation of the negative results obtained by Agger
et al. (1993). This late rise in local IgM raises the possibility that this response may be involved in the reduced worm burden observed in E. caproni-infected mice from 8 to 10 wpi (Odaibo et al., 1988). The IgM class is a major complement fixing antibody and complement has been suggested as one of the potential effector immune mechanisms against E. caproni (Simonsen and Andersen, 1986). Although this possibility cannot be disregarded completely, the lack of intestinal IgM response in rats and the earlier worm rejection in this host suggest that the role of local IgM response in parasite clearance could be considered only as secondary and other effector mechanisms should be implicated. The kinetics of total specific IgG observed in the serum of rats and mice are consistent with earlier studies (Agger et al., 1993; Graczyk and Fried, 1994; Toledo et al., 2004b,2005). The serum IgG response was markedly greater in mice than in rats probably in relation to a greater intestinal antigen uptake in relation to a higher local inflammation (Toledo et al., 2006a). Moreover, the kinetics of serum IgG1 and IgG2a were also different
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between both host species. Our results suggest a markedly biased response toward a Th2 phenotype and characterized by a strong production of IgG1 from 3 wpi and beyond. In rats an initial dominance of IgG2a response was observed. However, slight increase of IgG1 was detected from 7 wpi suggesting that early expulsion of E. caproni might be associated with balanced Th1/Th2 systemic responses. Brunet et al. (2000) analysed the IgG1 and IgG2a profiles in the serum of E. caproniinfected mice during the first 23 dpi and found a weak dominance of IgG2a on the last day of their experiment. These authors suggested that the development of chronic E. caproni infections was associated with Th1 responses. The discrepancies with respect to our results could be related to different methods used in each study. Brunet et al. (2000) only studied the initial phases of the infection. Our results show that during these phases the levels of IgG1 and IgG2a are low and no marked differences between both subclasses are established. However, at 3 wpi and beyond a progressive increase of IgG1 levels occurs. In fact, Brunet et al. (2000) did not detect changes in CD4+ and CD8+ cell populations and similar increases in both Th1 (IFN-c) and Th2 (IL-4) cytokines were observed during this initial period of the infection. In contrast to earlier studies, our results indicate that the development of long-lasting E. caproni infections is associated with dominance of systemic Th2 responses with high levels of serum IgG1 subclass. The IgG1 and IgG2a intestinal responses in mice and rats were slower and of lesser intensity than in serum. In mice, increases in both IgG1 and IgG2a subclasses were observed which could reflect a balanced Th1/Th2 local response. In contrast, rats only developed IgG2a response which suggests that local cellular responses could be of great importance in the parasite expulsion as previously suggested (Toledo et al., 2006a). Interestingly, the worm expulsion coincides with the pike of IgG2a observed in rats at 7 8 wpi. Although little is known about the mechanisms operating in natural hosts of E. caproni, cellular mechanisms could be of great importance in determining the course of the infection. Circulating and secretory IgA responses have been related to expulsion of intestinal protozoan and helminth parasite (Ben-Smith et al., 1999; Langford et al., 2002; Miquel et al., 2005). However, our results suggest that increases of IgA are not sufficient for E. caproni rejection. The infection in mice is able to stimulate high and rapid IgA antibody responses at both systemic and local levels although the survival of E. caproni is higher than in rats that do not exhibit IgA response. The induction of IgA response in mice might be the consequence of IL-6 production in response to exposure to E. caproni antigens, a Th2 cytokine that has been implicated in the switching to IgA production (Mazanec et al., 1993; Goodrich and McGee, 1999). In this context, the dominance of the Th2 responses observed in the serum of mice could explain the high IgA reactivity against E. caproni.
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Echinostoma caproni adults are attached to the host intestinal mucosa. Therefore, effective host defense against this digenean requires effector mechanisms that are active at the host mucosa. Our study suggests that the development of long-lasting E. caproni infection is related with dominance of Th2 systemic responses that appear not to be effective against the parasite. However, early expulsion of these worms from rodent hosts could be related to the generation of local Th1 responses involving cellular mechanisms. Acknowledgements The study was supported by the projects CGL200502321/BOS from the Ministerio de Educacio´n y Ciencia (Spain), GV04B/107 and GV05/039 from the Conselleria d’Empresa, Universitat I Cie`ncia de la Generalitat Valenciana (Spain) and the Project UV-AE-20050201 de la Universitat de Vale`ncia (Spain). This work has been carried out while the first author (J.S.) was recipient of a pre-doctoral fellowship from the Ministerio de Educacio´n y Ciencia, Madrid (Spain). This research complies with the current laws for animal health research in Spain. References Agger, M.K., Simonsen, P.E., Vennervald, B.J., 1993. The antibody response in serum, intestinal wall and intestinal lumen of NMRI mice infected with Echinostoma caproni. Journal of Helminthology 67, 169–178. Andresen, K., Simonsen, P.E., Andersen, B.J., Birch-Andersen, A., 1989. Echinostoma caproni in mice, shedding of antigens from the surface of an intestinal trematode. International Journal for Parasitology 19, 111–118. Ben-Smith, A., Wahid, F.N., Lammas, D.A., Behnke, J.M., 1999. The relationships between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection. Parasite Immunology 21, 383–395. Brunet, L.R., Joseph, S., Dunne, D.W., Fried, B., 2000. Immune responses during the acute stages of infection with the intestinal trematode Echinostoma caproni. Parasitology 120, 565–571. Christensen, N.Ø., Simonsen, P., Odaibo, A.B., Mahler, H., 1990. Establishment, survival and fecundity in Echinostoma caproni (Trematoda) infections in hamsters and jirds. Journal of the Helminthological Society of Washington 57, 104–107. Dea-Ayuela, M.A., Martı´nez-Ferna´ndez, A.R., Bola´s-Ferna´ndez, F., 2000. Comparison of IgG3 responses to carbohydrates following mouse immunization with six species of Trichinella. Journal of Helminthology 74, 215–223. Doumas, B., Watson, W., Biggs, H., 1971. Albumin standards and the measurement of serum albumin with bromocresol green. Clinica Chimica Acta 31, 87–96. Drabek, D., Raguz, S., De Wit, T.P., Dingjan, G.M., Savelkoul, H.F., Grosveld, F., Hendriks, R.W., 1997. Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under the control of the class II major histocompatibility complex Ea locus control region. Proceedings of the National Academy of Sciences 94, 610–615. Fried, B., Graczyk, T.K., 2004. Recent advances in the biology of Echinostoma species in the ‘‘revolutum’’ group. Advances in Parasitology 58, 139–195. Fried, B., Huffman, J.E., 1996. The biology of the intestinal trematode Echinostoma caproni. Advances in Parasitology 38, 311–368.
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