Parasite Immunology, 2014, 36, 522–530

DOI: 10.1111/pim.12125

Enhanced protection against Clonorchis sinensis induced by co-infection with Trichinella spiralis in rats K.-B. CHU,1 S.-S. KIM,1 S.-H. LEE,1 H.-S. LEE,2 K.-H. JOO,2 J.-H. LEE,3 Y.-S. LEE,4 S. ZHENG5 & F.-S. QUAN1,5 Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea, 2Department of Parasitology, Korea University College of Medicine, Seoul, Korea, 3Department of Pathology, Kyung Hee University Medical Center, Seoul, Korea, 4Korea Bioredginseng Co., LTD, Kumsan, South Korea, 5Department of parasitology, College of Medicine, Yanbian University, Yanji City, China 1

SUMMARY Although co-infection with multiple parasites is a frequent occurrence, changes in the humoral immune response against a pre-existing parasite induced as a result of a subsequent parasitic infection remain undetermined. Here, we utilized enzyme-linked immunosorbent assay (ELISA) to investigate antibody responses, cytokine production and enhanced resistance in Clonorchis sinensis-infected rats (Sprague–Dawley) upon Trichinella spiralis infection. Higher levels of C. sinensis-specific IgG and IgA were elicited upon T. spiralis infection, and these levels remained higher than in rats infected with C. sinensis alone. Upon subsequent infection with T. spiralis, IgG antibodies against C. sinensis appeared to be rapidly boosted at day 3, and IgA antibodies were boosted at day 7. Challenge infection of C. sinensis-infected rats with T. spiralis induced substantial mucosal IgG and IgA responses in the liver and intestine and increases in antibody-secreting plasma cells in the spleen and bone marrow. Subsequent infection also appeared to confer effective control of liver C. sinensis loads, resulting in enhanced resistance. Memory B cells generated in response to C. sinensis infection were rapidly amplified into antibody-secreting cells upon T. spiralis infection. These results indicate that enhanced C. sinensis clearance induced by co-infection is associated with systemic and mucosal IgG and IgA responses. Keywords Clonorchis sinensis, co-infection, helminth, IgA, IgG, Trichinella spiralis

Correspondence: Fu-Shi Quan, Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul 130-701, Korea (e-mail: [email protected]). Received: 4 February 2014 Accepted for publication: 18 June 2014

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INTRODUCTION Clonorchis sinensis, a parasitic trematode and the causative agent of clonorchiasis and human cholangitis, is widely distributed throughout eastern Asia, with a particularly high prevalence in China, Korea and Japan (1, 2). Human cases of clonorchiasis arise as a result of consuming undercooked fish containing C. sinensis metacercariae (3). Upon reaching the small intestine, the metacercariae exist and migrate towards the bile duct, causing obstruction of the bile duct and other diseases including bacterial infections, inflammation, periductal fibrosis, hyperplasia and cholangiocarcinoma (4, 5). Trichinellosis, another foodborne zoonotic disease with a worldwide distribution, is caused by consuming undercooked pork contaminated with Trichinella spiralis larvae (6, 7). T. spiralis has three different life stages in a single host, including an intestinal, a disseminated and a skeletal muscle stage. T. spiralis, upon reaching the hosts intestinal tract, invades the epithelial cells within the intestines and subsequently disseminates into the hosts skeletal muscles (8). At the beginning of the muscle phase, severe muscle pains can last until approximately day 50 of the infection. Different disorders may result from the dissemination of newborn larvae throughout the organism via blood and lymph vessels, resulting in damage to many different organs and tissues. The presence of worms in muscle tissue can also cause symptoms such as difficulty breathing, heart damage and various nervous disorders, eventually leading to death due to heart failure, respiratory complications or kidney malfunction (9). Both C. sinensis and T. spiralis are important parasites in many Asian countries, and emerging evidence suggests that infection with one parasite can significantly influence the outcome of a secondary parasitic infection (10). Although parasite co-infections are quite common worldwide, there is little information available about the © 2014 John Wiley & Sons Ltd

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impact of multiple parasite infections on parasite-specific immune responses (10). Co-infections result in differences in the induction of immune responses compared to infection with a single parasite (11). A shared novel antigen in Schistosoma mansoni and Plasmodium falciparum has been identified (12). Consequently, S. mansoni-infected mice produce antibodies that cross-react with the glycoproteins (glycan epitopes) of the helminth Trichinella spiralis (13, 14). Most co-infection studies have focused on resistance against subsequent parasitic infections, and we know of no previous study regarding the enhanced humoral immune responses against a pre-existing parasite induced as a result of a subsequent parasitic infection. In this study, we evaluated resistance against pre-existing C. sinensis in rats upon T. spiralis challenge infection. We focused on the resistance-related humoral IgG and IgA antibody responses that were induced upon secondary infection. We found that memory B-cell responses specific to C. sinensis were induced in the bone marrow and spleen. Our results indicate that T. spiralis challenge, in the context of a pre-existing C. sinensis infection, induced enhanced systemic and mucosal antibody responses against C. sinensis after 1 month. This enhanced response may contribute to a reduction in C. sinensis worm burden.

MATERIALS AND METHODS Animals and parasites Sprague–Dawley (SD) rats (females, 8 weeks old) and New Zealand white rabbits (male, 2–4 month old) were purchased from Samyook Animal Center, Osan City, Kyonggi-do, Korea. Adult SD rats were used for in vivo maintenance of the Trichinella spiralis Korean isolate. White rabbits were infected with Clonorchis sinensis metacercariae to generate C. sinensis adult worms. Trichinella spiralis larvae and C. sinensis metacercariae were collected from rats and from the freshwater fish Pseudorasbova parva, respectively, by digesting muscles with pepsin-HCl, followed by filtration through layers of gauze. All animal experiments and husbandry involved in the studies presented in this manuscript were conducted under the guidelines of the Kyung Hee University IACUC. Kyung Hee IACUC operates under the National Veterinary Research and Quarantine Service (NVRQS) and regulations of the World Organization for Animal Health (WOAH).

Cross-reactivity induced by helminth co-infection

RPMI 1640 medium (Welgene, Daegu, Korea) supplemented with penicillin, streptomycin and Fungizone (amphotericin B) (Invitrogen, Carlsbad, CA, USA) for 2 weeks at 37°C in the presence of 5% CO2. Culture medium was collected, and new medium was added daily. The collected medium was centrifuged at 4°C and 1000 rpm for 30 min, and the supernatants were lyophilized at 20°C. Protein concentration (8 mg/mL) was determined using the DC Protein Assay kit (Bio-Rad Lab, Hercules, CA, USA), and samples were stored at 70°C until use.

Boost effect and cross-reactivity between C. sinensis and T. spiralis To determine the boost effect, Sprague–Dawley (SD) rats (female, 8 wk old, 6 rats) were infected with 50 C. sinensis metacercariae at day 0 (prime), month 1 (1st boost) and month 2 (2nd boost). Sera were collected at day 0 and week 3 after prime, 1st boost and 2nd boost. To compare antibody responses after a single exposure to C. sinensis with those after receiving 1st boost, sera were collected at day 0, weeks 2, 4 and 6 after prime and 1st boost. Sera from rats after prime infection served as controls. Rats were also only infected with 1000 T. spiralis at day 0 (prime). To determine the antibody cross-reactivity between C. sinensis and T. spiralis, C. sinensis or T. spiralis-infected sera or liver were collected from rats infected with 50 C. sinensis metacercariae or 1000 muscle larvae of T. spiralis at week 4. Livers were homogenized in PBS, and samples were collected using a cell strainer. ELISA was performed to determine boost effects as described previously (4). To measure parasite-specific antibodies, 100 lL ES antigen (4 lg/mL) in coating buffer (01 M sodium carbonate, pH 95) was coated onto 96-well microtitre plate at 4°C overnight. ELISA and Western blot were used to determine whether T. spiralis antibody crossreacted with C. sinensis ES Ag. Reactivity to C. sinensis ES Ag was analysed by probing with anti-C. sinensis or anti-T. spiralis antibodies and HRP-conjugated anti-rat IgG antibody (AbD Serotec, Kidlington, UK). AntiC. sinensis and anti-T. spiralis antibodies were produced in rats infected with C. sinensis or T. spiralis at week 4 after prime, respectively.

C. sinensis infection and subsequent infection with T. spiralis in rats

Clonorchis sinensis were collected from rabbit liver, and the excretory–secretory antigen (ES Ag) of C. sinensis was obtained by culturing living C. sinensis adults (500 worms) in

Sprague–Dawley (SD) rats (females, 8 weeks old) were used. Groups of rats were previously infected with 50 C. sinensis metacercariae. After 1 month, groups infected with C. sinensis were divided into two groups. One group was subsequently infected with T. spiralis (CS+TS group, 6

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rats). Another group infected with only C. sinensis was used as a single-infection control (CS group, 6 rats). Six na€ıve rats were used as uninfected control. One month after T. spiralis infection, C. sinensis adult worms were collected from the bile ducts of all rats.

Serum antibody responses Serum samples were collected from rats at the ophthalmic venous plexus at days 3, 7, 14 and 30 after T. spiralis infection (CS+TS) or C. sinensis single infection (CS). C. sinensis-specific total IgG and IgA antibody responses were determined at days 3, 7, 14 and 30 after infection in the na€ıve, C. sinensis single infection (CS) and C. sinensis and T. spiralis co-infection (CS+TS) groups by ELISA. Plates (Nunc MaxiSorpâ flat-bottom 96-well plate) were coated with 100 lL of C. sinensis ES Ag (4 lg/mL), and HRP-conjugated anti-IgG (Bio-Rad) and anti-IgA (BioRad) were used as secondary antibodies, as described previously (15).

IgG and IgA antibody response profiles in the intestine and liver upon T. spiralis infection Liver and intestine were collected from co-infected, C. sinensis single-infection control and na€ıve control rats 1 month after subsequent infection with T. spiralis. Individual livers were collected from rats and homogenized in PBS. Each individual liver sample was applied to a mesh cell strainer. The strained sample was centrifuged at 500 g for 10 min, and the supernatant was stored at 70°C until use. Individual rat small intestines were collected from a location 10 cm beneath the stomach. The collected intestine was incubated in 085% saline at 37°C for 1 h. The intestinal mucus was collected and centrifuged at 2000 rpm for 10 min. The supernatant was stored at 70°C until use.

Parasite Immunology

the coated antigens were determined as previously described (16).

Cytokine response Individual rat spleens were collected month 1 after challenge from the CS+TS, CS and na€ıve groups. Single-cell suspensions were prepared from each spleen. Cells were resuspended in RPMI 1640 supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, 25 mM HEPES, 05 M 2-mercaptoethanol, 1% sodium pyruvate, penicillin and streptomycin. Cells (05 9 106/well) were incubated in 96-well flat culture plates for 3 days at 37°C in the presence of 5% CO2. Cells in 100 lL of RPMI-1640 were stimulated with 100 lL of 10 lg/mL ES antigen. For the cytokine assay, supernatants of spleen cell cultures were collected from each well by centrifugation and stored at 70°C until use. OptEIA sets (BD/PharMingen, San Jose, CA, USA) were used to determine IFN-c, IL-2, IL-4 and IL-10 concentrations in culture supernatants following the manufacturers procedures.

Statistical analysis The C. sinensis adult worm burden and serum antibody levels were recorded for each individual. Every assay was performed using at least three replicate samples, from which the arithmetic mean and standard error of the mean were calculated. A two-tailed Students t-test was performed when comparing two different conditions. When comparing three conditions, a one-way analysis of variance (ANOVA) was performed. A value of P < 005 was considered significant.

RESULTS

Analysis of antibody-secreting cell response in vitro

C. sinensis antibody is boosted by subsequent infection with T. spiralis, and T. spiralis antibody is cross-reactive to C. sinensis ES antigens

To detect C. sinensis-specific antibody-producing cells, C. sinensis ES antigen (4 lg/mL, 100 lL) was used to coat 96-well culture plates (Costar). Freshly isolated cells from the bone marrow and spleen (1 9 106 cells/well) were added to each well and incubated for 4 days at 37°C in the presence of 5% CO2. After removing cells from the culture plates, HRP-conjugated secondary goat anti-rat antibodies (Bio-Rad) were added. As a measure of HRP activity, the substrate o-phenylenediamine (Zymed, San Francisco, CA, USA) was used, and the optical density was measured at 490 nm. The levels of parasite-specific antibodies secreted into the culture media and bound to

To determine whether antibody production was enhanced by boosting, rats were infected with C. sinensis metacercariae three times and antibody responses were determined after the prime, 1st boost and 2nd boost. IgG antibody responses were significantly enhanced after the 1st boost and not increased after the 2nd boost (Figure 1a). Higher levels of IgG were found in boosted rats compared to those receiving a single C. sinensis infection (Figure 1b). These data provided the rationale for determining whether the boost effect can also be induced by subsequent infection with T. spiralis, particularly as common antigens are presented between C. sinensis and T. spiralis (Figure 2a).

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Figure 1 Antibody responses in rats infected with C. sinensis. Rats were infected with C. sinensis metacercariae three times, and antibody levels were determined after the prime, 1st boost and 2nd boost infections (a). 500x: Sera used in determining antibody responses were diluted 500-fold. 50000x: Sera used in determining antibody response were diluted 50 000-fold. Antibody responses in rats after boosting were compared with those after a single exposure to C. sinensis (b). Sera were collected from rats: na€ıve, primary infected (CS) and after the first boosting (CS/CS) on weeks 2 (W2), 4 (W4) and 6 (W6). Sera used were diluted 100-fold.

reactive to T. spiralis (Figure 2b, c). To determine the level of cross-reactivity between C. sinensis and T. spiralis, Western blot analyses were performed using C. sinensis ES antigen, and the reactivity of C. sinensis ES Ag was investigated with either anti-C. sinensis or anti-T. spiralis antibodies. Strong cross-reactivity was observed, resulting in

Antibody responses to C. sinensis ES antigen from rats infected with either C. sinensis or T. spiralis alone were analysed to determine cross-reactivity. Cross-reactive IgG antibodies against C. sinensis or T. spiralis were found in the sera and liver. Higher levels of antibody reactive to C. sinensis were present as compared to levels of antibody

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Figure 2 Cross-reactivity between C. sinensis and T. spiralis. (a) SDS-PAGE and Western blotting were used to determine the crossreactivity between C. sinensis and T. spiralis. Reactivity to C. sinensis ES Ag was analysed using anti-C. sinensis (500-fold diluted) or antiT. spiralis antibodies (500-fold diluted) and HRP-conjugated anti-rat IgG antibody. Sera from rats infected with C. sinensis or T. spiralis at week 4 after prime were used as a source of parasite-specific antibodies. M: marker; lanes 1, 2, 3, 4 and 5, 6, 7, 8 represent 5, 10, 15 and 20 lg of C. sinensis ES Ag, respectively. Lanes N1 and N2 represent 10 lg and 20 lg of C. sinensis ES Ag. Lanes 1, 2, 3 and 4 were incubated with serum obtained from rats infected with C. sinensis, while lanes 5, 6, 7 and 8 were incubated with serum obtained from rats infected with T. spiralis. Lanes N1 and N2 were incubated with serum of uninfected rats as controls. (b, c) The reactivity of C. sinensis ES Ag with specific antibodies present in the sera of C. sinensis or T. spiralis-infected animals. C. sinensis ES antigen is either reactive to C. sinensis antibody (CS infection) or to T. spiralis antibody (TS infection) in sera (b) and liver (c). Sera were collected from rats: na€ıve, primary infected with C. sinensis or T. spiralis on weeks 1 (W1), 2 (W2) and 4 (W4). Sera used were diluted 100-fold. 5x: Liver sample was diluted fivefold, 50x: Liver sample was diluted 50-fold. © 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 522–530

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High levels of C. sinensis-specific serum IgG and IgA antibodies are induced upon subsequent infection with T. spiralis To determine antibody response profiles, groups of rats were initially infected with C. sinensis. At day 30, these rats were subsequently infected with T. spiralis. IgG and IgA antibody responses reactive to C. sinensis ES antigen were evaluated at days 3, 7, 14 and 30 following the initial C. sinensis infection and at days 33, 37, 44 and 60 following the subsequent infection with T. spiralis. At day 33, significantly higher levels of C. sinensis-specific IgG antibody were detected in the subsequently T. spiralis-infected rats as compared to in rats infected only with C. sinensis (CS) and antibody levels remained high until day 60 (Figure 3a). At day 44, the 14th day after subsequent infection T. spiralis, significantly higher levels of C. sinensis-specific IgA antibodies were detected (Figure 3b) compared with the levels seen in the CS control group. These results indicate that IgG and IgA antibodies were rapidly boosted by subsequent infection with T. spiralis.

Subsequent infection with T. spiralis induces enhanced mucosal IgG and IgA antibodies against C. sinensis in intestine and liver

IgG specific to C. sinensis ES antigen (OD)

As both C. sinensis and T. spiralis infections can be established by oral delivery, infection is believed to occur via

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the mucosal route. C. sinensis resides in the liver of humans where it is found mainly in the common bile duct. T. spiralis resides in the human intestine early in its life cycle. Thus, we measured C. sinensis-specific IgG and IgA antibody responses in mucosal sites in the liver and intestine. In C. sinensis-infected rats co-infected with T. spiralis, higher levels of IgG and IgA antibodies were seen in the intestine (Figure 4a, b; *P < 005, **P < 001) and liver (Figure 4c, d; *P < 005, **P < 001) than were seen in the intestine and liver of unchallenged animals. Oral administration of C. sinensis induced mucosal IgG and IgA antibodies, and this response was significantly enhanced upon mucosal challenge with T. spiralis. Thus, enhanced mucosal IgG and IgA antibodies might be involved in resistance to C. sinensis induced by subsequent infection with T. spiralis.

Subsequent infection with T. spiralis induces a marked anti-C. sinensis antibody-secreting cell response To determine antibody-secreting cell responses after subsequent infection with T. spiralis, bone marrow and spleen cells were collected from rats and subjected to in vitro culture. We found that significantly higher levels of IgG antibodies specific to C. sinensis were secreted into culture supernatants by bone marrow and spleen cells of T. spiralis-co-infected rats than from cells derived from rats infected only with C. sinensis (Figure 5a, c; *P < 005, **P < 001). We also evaluated the IgA antibodies in the same culture supernatants (Figure 5b, d; *P < 005, **P < 001). Significant levels of IgA antibodies specific to C. sinensis were detected after 4 days of culture. Taken together, these results indicate that C. sinensis infection can systemically generate memory B cells that have the

IgA specific to C. sinensis ES Ag (OD)

bands with molecular weights of 43, 80, 95 and 130 KDa (Figure 2a). This indicates that C. sinensis and T. spiralis share some antigens. This may contribute to the rapid antibody response seen after T. spiralis challenge infection in C. sinensis-infected rats, as described above.

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Figure 3 C. sinensis-specific IgG and IgA antibody response in serum. Groups of rats (n = 12) were infected with 50 C. sinensis metacercariae, and then 6 rats were infected with 1000 T. spiralis larvae 1 month later (CS+TS). Rats receiving C. sinensis single infections (CS) were used as controls. Blood samples were collected at days 3, 7, 14 and 30 after C. sinensis infection and at days 3 (33), 7 (37), 14 (44) and 30 (60) after T. spiralis challenge infection. C. sinensis-specific IgG (a) and IgA (b) antibody responses were determined. As controls, blood samples from the CS group were also collected at the same time points listed above. Three independent experiments were performed, and the data shown represent the average of several independent experiments. Shown is the average  standard error of the mean from n = 6 rats.

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Figure 4 IgG and IgA responses in liver and intestine. Groups of rats (n = 6) were infected with C. sinensis, and 1 month later, CS+TS rats were co-infected infected with T. spiralis. Na€ıve rats were not infected at all. Liver samples were collected at day 30 post-challenge infection (CS+TS group) and after single infection (CS group). Mucosal IgG (a) and IgA (b) antibody responses in the liver were determined. For mucosal IgG, significant differences were detected between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (**P < 001), and between the CS+TS and na€ıve groups (**P < 001). For IgA, significant differences were detected between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (*P < 005) and between the CS+TS and na€ıve groups (**P < 001). Intestine samples were collected at day 30 after T. spiralis infection (CS+TS group) and at day 60 after single infection (CS group). Mucosal IgG (c) and IgA (d) antibody responses in the intestine were determined. For mucosal IgG, significant differences were detected between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (**P < 001) and between the CS+TS and na€ıve groups (**P < 001). For IgA, significant differences were found between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (*P < 005) and between the CS+TS and na€ıve groups (**P < 001). Shown is the average  standard error of the mean from n = 6 rats.

capacity to rapidly differentiate into antibody-secreting cells upon subsequent infection with T. spiralis.

C. sinensis worm loads are significantly reduced after subsequent infection with T. spiralis To assess the protection against C. sinensis conferred by subsequent infection with T. spiralis, we determined the worm loads in liver ducts on day 30. After T. spiralis infection, the rats showed significantly lower liver worm burdens (27%) than those seen in rats infected with only C. sinensis (Figure. 6). The results indicate that subsequent infection with T. spiralis can induce protective immune responses which effectively reduce C. sinensis worm burden in the liver.

IL-10 were produced following C. sinensis ES antigen stimulation in both the CS+TS and CS groups relative to the levels seen in na€ıve rats (Figure. 7a, b, d; *P < 005). However, no significant differences were found between the CS+TS and CS groups, indicating that cytokine responses may not enhanced by T. spiralis challenge during co-infection.

DISCUSSION

To evaluate cytokine production, splenocytes were harvested 1 month after subsequent infection. The levels of production of IFN-gamma, IL-2, IL-4 and IL-10 from cytokine-secreting cells were determined. As shown in Figure. 7, significantly higher levels of IFN-gamma, IL-2 and

Shared antigenic components and induced cross-reactivity have been previously identified among parasites (11, 12, 14, 17–19). In parasite co-infections, primary infections can result in resistance to secondary parasite infections (10, 20). These studies mostly demonstrated cross-protection against subsequent parasite infections. However, in the present study, we focused on and investigated immunity against a pre-existing C. sinensis parasite induced by subsequent T. spiralis infection. Our study resulted in the novel finding that high levels of antibodies against C. sinensis were induced upon T. spiralis infection, resulting in the reduction of C. sinensis worm burden in C. sinensis/ T. spiralis-co-infected animals.

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Figure 5 IgG and IgA antibody-secreting cells in spleen and bone marrow. Groups of rats (n = 6) were infected with C. sinensis, and the CS+TS group was infected with T. spiralis 1 month later. Na€ıve rats were not infected at all. Spleen (a, b) and bone marrow (c, d) samples were collected from all groups at day 30 after T. spiralis infection. Significant differences in the IgG response were detected between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (*P < 005) and between the CS+TS and na€ıve groups (**P < 001) (a, c). Significant differences in the IgA response were detected between the CS+TS and CS groups (*P < 005), between the CS and na€ıve groups (*P < 005) and between the CS+TS and na€ıve groups (*P < 005) (b, d). Shown is the average  standard error of the mean from n = 6 rats.

infection in the context of C. sinensis/T. spiralis co-infections is likely different from the mechanisms studied in our current report. More comprehensive studies will be needed to fully evaluate these differences and elucidate the mechanisms involved. Interestingly, we observed that higher levels of mucosal IgG and IgA antibody against C. sinensis ES antigen in

Figure 6 Protection against C. sinensis induced by subsequent infection with T. spiralis in rats. Groups of rats (n = 6) were infected with C. sinensis, and the CS+TS group was subsequently infected with T. spiralis 1 month later. Rats were sacrificed at day 30 after T. spiralis infection. C. sinensis worms were collected and counted. A significant difference in worm burden was found between the CS+TS and CS groups (*P < 005).

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No. of C. sinensis recovered

We observed that high levels of serum and mucosal IgG and IgA antibodies were induced against C. sinensis ES antigen upon T. spiralis challenge. IgG and IgA antibodies against C. sinensis appear to be rapidly boosted at days 3 (day 33 post-infection) and 21 (day 51 post-infection), respectively. Antibody responses were significantly enhanced against C. sinensis and T. spiralis in C. sinensisinfected rats, indicating that antigens shared between C. sinensis and T. spiralis played a role in this response. Our Western blot data demonstrated that antigens from C. sinensis reacted to both C. sinensis- and T. spiralisinfected rat antibodies. Thus, in our study, antigenic proteins derived from a T. spiralis challenge infection boosted antibody responses against a pre-existing C. sinensis infection. In contrast, we found that no enhanced antibody response was induced against T. spiralis in C. sinensis/ T. spiralis-co-infected animals (data not shown). This indicates that additional protective immune mechanisms may be involved in addition to humoral antibody responses. Recently, it was reported that severe enteric histopathological changes and increased cytokine levels reduce T. spiralis worm burden in mice pre-infected with C. sinensis in the context of C. sinensis/T. spiralis co-infection (10). The underlying mechanism responsible for inducing protection against pre-existing C. sinensis or subsequent T. spiralis

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Figure 7 Cytokine response. Groups of rats (n = 6) were infected with C. sinensis, and the CS+TS group was subsequently infected with T. spiralis 1 month later. Rats were sacrificed at day 30 after T. spiralis infection. Splenocytes were cultured in RPMI-1640 media for 3 days, and IFN-c, IL-2, IL-4 and IL-10 were measured in the supernatants. Significant differences in cytokine responses (IFN-c, IL-2 and IL-10) were found between na€ıve and CS groups (*P < 005), and between na€ıve and CS+TS groups (*P < 005).

the liver and in intestinal mucosa samples were induced after T. spiralis challenge infection. Both the liver and intestine are important organs where C. sinensis and T. spiralis reside or enter the body. It is possible to achieve enhanced mucosal immune responses at mucosal surface liver ducts and intestines through oral infection with T. spiralis in rats orally pre-infected with C. sinensis. This phenomenon involves gut-associated lymphoid tissues, such as the Peyers patches and mesenteric lymph nodes, and leads to the dissemination of antigen-sensitized immune cells to mucosal tissues via the efferent lymphatics (21). In particular, mucosal IgA antibodies in the bile ducts of liver are known to be associated with the resistance against C. sinensis in rats (22). Mucosal antibodies are produced by B lymphocytes adjacent to the mucosal cells, transported through the cell interior and released as secretions from the cells. In addition, a parasite-specific IgA response was shown to play a role in human bancroftian filariasis (23). The IgA response to T. spiralis newborn larva is stage specific (24). These results indicate that oral T. spiralis challenge infection may boost IgA antibody responses against C. sinensis ES antigen as common epitopes are presented by both C. sinensis and T. spiralis. Infection or vaccination can generate long-lived memory B cells that can rapidly differentiate into antibody-secreting plasma cells upon exposure to antigens (25). We found that high levels of antibody-secreting cells (ASCs) derived from the spleen and bone marrow were induced by C. sinensis/T. spiralis co-infection. We also observed

that IgG- or IgA-producing ASCs neutralized C. sinensis ES antigens (data not shown). As antibody-secreting plasma cells in the spleen and bone marrow are long-lived and responsible for maintaining serum antibodies, subsequent T. spiralis infection induced an enhanced and rapid boost in antibody levels (26, 27). Primary infection with C. sinensis triggered the development of memory B cells and, because subsequent T. spiralis infection containing antigens was reactive to C. sinensis, caused memory B cells to differentiate into ASCs. As anticipated, this eventually functioned as a protective mechanism and lowered the parasite load within the host. Our data indicate that no significant increase in cytokine levels was detectable in co-infections relative to the levels in C. sinensis single infection, indicating that cytokines may not be involved in the T. spiralis infectioninduced changes in the immune response to pre-existing C. sinensis infection. In contrast, a mixed Th1- and Th2type immune response is induced in humans concurrently infected with Necator americanus and Oesophagostomum bifurcum (11). This may be due to differences in the parasite species used in the studies and also to differences in the hosts and the duration of the infections. A more detailed investigation will be required to elucidate the complex immune mechanisms resulting in these different patterns of immune response. The present findings demonstrate that protection against a pre-existing C. sinensis infection in rats was induced by subsequent infection with T. spiralis. Increases in systemic and mucosal IgG and IgA specific to C. sinensis upon

© 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 522–530

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subsequent infection with T. spiralis may contribute to protection against pre-existing C. sinensis infection.

funded by the MEST and ICT and future Planning and by Korea research foundation grant No. 20120007792.

ACKNOWLEDGEMENTS This work (NRF-2014R1A2A2A01004899) was supported by Mid-career Researcher Program through NRF grant

DISCLOSURES The authors declare that there are no conflicts of interest.

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© 2014 John Wiley & Sons Ltd, Parasite Immunology, 36, 522–530

Enhanced protection against Clonorchis sinensis induced by co-infection with Trichinella spiralis in rats.

Although co-infection with multiple parasites is a frequent occurrence, changes in the humoral immune response against a pre-existing parasite induced...
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