Trematodes coupled with neonicotinoids: effects on blood cell profiles of a model amphibian.

Habitat loss, climate change, environmental contaminants, and parasites and pathogens are among the main factors thought to act singly or together in causing amphibian declines. We tested for combined effects of neonicotinoid pesticides and parasites (versus parasites-only) on mortality, growth, and white blood cell profiles of a model amphibian: the northern leopard frog (Rana pipiens). We first exposed infectious stages of frog trematodes (cercariae of Echinostoma spp.) to low and high concentrations of thiamethoxam or clothianidin versus water-only controls. There were no differences in survival of trematode cercariae between treatments. For the main experiment, we exposed tadpoles to clean water versus high concentrations of clothianidin or thiamethoxam for 2 weeks and added trematode cercariae to all tanks after 1 week. Exposure of tadpoles and parasites to high concentrations of thiamethoxam or clothianidin did not affect parasite infection success. Tadpole survival was not different between treatments before or after parasite addition and there were no significant differences in tadpole snout-to-vent lengths or developmental stages between treatments. Tadpoles exposed to thiamethoxam + parasites had smaller widths than parasite-only tadpoles, whereas tadpoles exposed to clothianidin + parasites had higher eosinophil to leukocyte ratios compared to parasite-only tadpoles. Tadpoles of both neonicotinoid + parasite treatments had significantly lower monocyte to leukocyte ratios relative to parasite-only tadpoles. High concentrations of neonicotinoid combined with parasites appear to influence tadpole immune function important for further defense against parasites and pathogens. This work highlights the need for more holistic approaches to ecotoxicity studies, using multiple stressors.

Ileal proteomic changes associated with IL-25-mediated resistance against intestinal trematode infections.

BACKGROUND: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been extensively used to investigate the factors that determine the rejection of intestinal helminths. In this sense, several studies have shown that IL-25 is critical for the development of resistance against E. caproni in mice. In fact, treatment of mice with recombinant IL-25 generates resistance against primary E. caproni infection. However, the mechanisms by which IL-25 induces resistance remain unknown. METHODS: To study the mechanisms responsible for resistance elicited by IL-25, we analyzed the ileal proteomic changes induced by IL-25 in mice and their potential role in resistance. To this purpose, we compared the protein expression profiles in the ileum of four experimental groups of mice: naïve controls; E. caproni-infected mice; rIL-25-treated mice; and rIL-25-treated mice exposed to E. caproni metacercariae. RESULTS: Quantitative comparison by 2D-DIGE showed significant changes in a total of 41 spots. Of these, 40 validated protein spots were identified by mass spectrometry corresponding to 24 proteins. CONCLUSIONS: Our results indicate that resistance to infection is associated with the maintenance of the intestinal epithelial homeostasis and the regulation of proliferation and cell death. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection and its transcendence in resistance.

Antibody trapping: A novel mechanism of parasite immune evasion by the trematode Echinostoma caproni.

BACKGROUND: Helminth infections are among the most prevalent neglected tropical diseases, causing an enormous impact in global health and the socioeconomic growth of developing countries. In this context, the study of helminth biology, with emphasis on host-parasite interactions, appears as a promising approach for developing new tools to prevent and control these infections. METHODS/PRINCIPAL FINDINGS: The role that antibody responses have on helminth infections is still not well understood. To go in depth into this issue, work on the intestinal helminth Echinostoma caproni (Trematoda: Echinostomatidae) has been undertaken. Adult parasites were recovered from infected mice and cultured in vitro. Double indirect immunofluorescence at increasing culture times was done to show that in vivo-bound surface antibodies become trapped within a layer of excretory/secretory products that covers the parasite. Entrapped antibodies are then degraded by parasite-derived proteases, since protease inhibitors prevent for antibody loss in culture. Electron microscopy and immunogold-labelling of secreted proteins provide evidence that this mechanism is consistent with tegument dynamics and ultrastructure, hence it is feasible to occur in vivo. Secretory vesicles discharge their content to the outside and released products are deposited over the parasite surface enabling antibody trapping. CONCLUSION/SIGNIFICANCE: At the site of infection, both parasite secretion and antibody binding occur simultaneously and constantly. The continuous entrapment of bound antibodies with newly secreted products may serve to minimize the deleterious effects of the antibody-mediated attack. This mechanism of immune evasion may aid to understand the limited effect that antibody responses have in helminth infections, and may contribute to the basis for vaccine development against these highly prevalent diseases.

Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus).

Many organisms face energetic trade-offs between defense against parasites and other host processes that may determine overall consequences of infection. These trade-offs may be particularly evident during unfavorable environmental conditions or energetically demanding life history stages. Amphibian metamorphosis, an ecologically important developmental period, is associated with drastic morphological and physiological changes and substantial energetic costs. Effects of the trematode parasite Echinostoma trivolvis have been documented during early amphibian development, but effects during later development and metamorphosis are largely unknown. Using a laboratory experiment, we examined the energetic costs of late development and metamorphosis coupled with E. trivolvis infection in wood frogs, Lithobates [=Rana] sylvaticus. Echinostoma infection intensity did not differ between tadpoles examined prior to and after completing metamorphosis, suggesting that metacercariae were retained through metamorphosis. Infection with E. trivolvis contributed to a slower growth rate and longer development period prior to the initiation of metamorphosis. In contrast, E. trivolvis infection did not affect energy expenditure during late development or metamorphosis. Possible explanations for these results include the presence of parasites not interfering with pronephros degradation during metamorphosis or the mesonephros compensating for any parasite damage. Overall, the energetic costs of metamorphosis for wood frogs were comparable to other species with similar life history traits, but differed from a species with a much shorter duration of metamorphic climax. Our findings contribute to understanding the possible role of energetic trade-offs between parasite defense and host processes by considering parasite infection with simultaneous energetic demands during a sensitive period of development.

Subcutaneous injection of exosomes reduces symptom severity and mortality induced by Echinostoma caproni infection in BALB/c mice.

Recent studies have shown the importance of exosomes in the host-parasite relationship. These vesicles are an important part of the excretory/secretory pathway for proteins with the potential to alter immune responses. Therefore, in the present study, we examined the immunomodulatory role of exosomes in BALB/c mice using Echinostoma caproni as an experimental model of intestinal helminth infection. For this purpose, BALB/c mice were injected twice s.c. with purified exosomes of E. caproni, followed by experimental infection. We report a delay in the development of the parasite in mice immunised with exosomes, a concomitant reduced symptom severity and increased survival upon infection. Immunisations with exosomes evoked systemic antibody responses with high levels of IgM and IgG. IgG1, IgG2b and IgG3 are the subtypes responsible for the IgG increase. These antibodies showed specific recognition of exosomal proteins, indicating that these vesicles carry specific antigens that are involved in the humoral response. The administration of exosomes induced an increase of IFN-γ, IL-4 and TGF-ß levels in the spleen of mice prior to infection. The subsequent infection with E. caproni resulted in a further increase of IL-4 and TGF-ß, together with an abrupt overproduction of IL-10, suggesting the development of a Th2/Treg immune response. Our results show that the administration of exosomes primes the immune response in the host, which in turn can contribute to tolerance of the invader, reducing the severity of clinical signs in E. caproni infection.

Resistance against Echinostoma caproni (Trematoda) secondary infections in mice is not dependent on the ileal protein production.

UNLABELLED: Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode, which has been widely employed to investigate the factors determining the rejection of intestinal helminths. Protein production patterns of intestinal epithelial cells are related to the infection-induced changes that determine the course of E. caproni infections. Herein, we compare the protein production profiles in the ileum of four experimental groups of mice: control; infected; dewormed and reinfected. Worm burdens were significantly lower in secondary infections, confirming the generation of partial resistance to homologous secondary infections in mice. However, quantitative comparison by 2D-DIGE showed that the protein production profile is similar in control and dewormed mice, and after primary and secondary E. caproni infections. These results showed that, unexpectedly, protein production changes in E. caproni infections are not responsible of resistance development. Fifty-one protein spots were differentially produced between control/treated and infected/reinfected mice and 37 of them were identified by mass spectrometry. The analysis of differentially abundant proteins indicate that cell metabolism and the regulation of proliferation and cell death are the most affected processes after primary and secondary E. caproni infections. These results provide new insights into the proteins involved in the regulation of tissue homeostasis after intestinal infection. SIGNIFICANCE: Intestinal helminthiases are highly prevalent parasitic infections with about 1 billion people infected worldwide. In this scenario, better understanding of host-parasite relationships is needed to elucidate the factors that determine intestinal helminth rejection. The intestinal trematode Echinostoma caproni has been broadly employed in this field, with resistance against secondary homologous infections reported in mice. In this paper, new insights are provided in the regulation of tissue homeostasis after intestinal infection. The unexpected lack of an altered pattern of ileal protein production associated to resistance development suggests that this resistance depends on rapid changes, affecting the early establishment of worms, rather than the activation of later effector mechanisms. These results may contribute to the development of new control tools for the management of these parasitic infections.

Differential expression and glycosylation of proteins in the rat ileal epithelium in response to Echinostoma caproni infection.

Echinostoma caproni (Trematoda: Echinostomatidae) is an intestinal trematode that has been used as experimental model to investigate the factors determining the expulsion of intestinal helminths. We analyze the changes in the protein expression and glycosylation induced by E. caproni in Wistar rat, a host of low compatibility in which the parasites are rapidly rejected. To determine the changes in protein expression, two-dimensional difference gel electrophoresis was employed using protein extracts from the intestine of naïve and infected rats. The patterns of glycosylation were analyzed by lectin blotting. Those spots showing differential expression or glycosylation were analyzed by mass spectrometry. A total of 33 protein spots differentially expressed were identified (26 were found to be over-expressed and 7 down-regulated). Moreover, E. caproni induced changes in the glycosylation status of 8 proteins that were successfully identified. Most of these proteins were related to the cytoskeleton and the maintenance of the functional integrity of the ileal epithelium. This suggests that the regeneration of the intestinal tissue is a major effector mechanism responsible for the early expulsion of this helminth. Furthermore, several proteins involved in the energy metabolism were also altered in the ileum of rats as a consequence of the E. caproni infection. BIOLOGICAL SIGNIFICANCE: Our analysis provides essential new insights in the factors determining the natural expulsion of intestinal parasitic helminths from their hosts. The results obtained contribute to a better understanding of the effective mechanisms involved in the defense against the intestinal helminths. The identification of proteins in the intestine that become modified in their expression or glycosylation in hosts in which the parasite is rapidly rejected may serve for the development of tools for the control of these infections.

Echinostoma revolutum: freshwater snails as the second intermediate hosts in Chiang Mai, Thailand.

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.

Echinostoma trivolvis (Digenea: Echinostomatidae) second intermediate host preference matches host suitability.

Many trematodes infect a single mollusk species as their first intermediate host, and then infect a variety of second intermediate host species. Determining the factors that shape host specificity is an important step towards understanding trematode infection dynamics. Toward this end, we studied two pond snails (Physa gyrina and Helisoma trivolvis) that can be infected as second intermediate hosts by the trematode Echinostoma trivolvis lineage a (ETa). We performed laboratory preference trials with ETa cercariae in the presence of both snail species and also characterized host suitability by quantifying encystment and excystment success for each host species alone. We tested the prediction that trematodes might preferentially infect species other than their obligate first intermediate host (in this case, H. trivolvis) as second intermediate hosts to avoid potentially greater host mortality associated with residing in first intermediate hosts. In our experiments, ETa had roughly equivalent encystment success in Helisoma and Physa snails, but greater excystment success in Physa, when offered each species in isolation. Also, the presence of the symbiotic oligochaete Chaetogaster limnaei in a subset of Helisoma snails reduced encystment success in those individuals. When both hosts were present, we found dramatically reduced infection prevalence and intensity in Helisoma-ETa cercariae strongly preferred Physa. Thus, the presence of either an alternative host, or a predator of free-living parasites, offered protection for Helisoma snails from E. trivolvis lineage a infection.

The influence of adult worm age and definitive host on the transmission of Echinostoma caproni: egg hatchability and miracidial infectivity.

We analysed the influence of different ages of Echinostoma caproni adult worms reared in different definitive host-species on egg hatchability and miracidial infectivity. Eggs obtained from 2-, 4- and 6-week-old adult worms collected from rats, hamsters and mice, hatched miracidia that were used to infect laboratory-reared specimens of Biomphalaria glabrata. Highest egg hatchability was obtained with eggs derived from hamsters and mice. Egg hatchability did not seem to be affected by the age of E. caproni adult worms. Miracidial infectivity was not affected by the definitive host species. However, miracidial infectivity was affected by the age of adult worms when they developed in the mice intestine. The long period of miracidial infectivity in hamsters revealed this definitive host as the most suitable for experimental development of the life cycle and parasite transmission.

Systems parasitology: effects of Fasciola hepatica on the neurochemical profile in the rat brain.

We characterize the integrated response of a rat host to the liver fluke Fasciola hepatica using a combination of (1)H nuclear magnetic resonance spectroscopic profiles (liver, kidney, intestine, brain, spleen, plasma, urine, feces) and multiplex cytokine markers of systemic inflammation. Multivariate mathematical models were built to describe the main features of the infection at the systems level. In addition to the expected modulation of hepatic choline and energy metabolism, we found significant perturbations of the nucleotide balance in the brain, together with increased plasma IL-13, suggesting a shift toward modulation of immune reactions to minimize inflammatory damage, which may favor the co-existence of the parasite in the host. Subsequent analysis of brain extracts from other trematode infection models (i.e. Schistosoma mansoni, and Echinostoma caproni) did not elicit a change in neural nucleotide levels, indicating that the neural effects of F. hepatica infection are specific. We propose that the topographically extended response to invasion of the host as characterized by the modulated global metabolic phenotype is stratified across several bio-organizational levels and reflects the direct manipulation of host-nucleotide balance.

Excretory/secretory proteome of the adult stage of Echinostoma caproni.

The excretory/secretory proteome of Echinostoma caproni (Trematoda: Echinostomatidae) adults collected from experimentally infected mice was investigated using a proteomic approach. We performed a shot-gun liquid chromatography/tandem mass spectrometry for the separation and identification of tryptic peptides from the excretory/secretory products of E. caproni adult worms. Database search was performed using MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems). A total of 39 parasite proteins were accurately identified. Strikingly, metabolic enzymes, and particularly glycolytic enzymes, constituted the largest protein family in the excretory/secretory proteome of E. caproni adult worms. Moreover, representative proteins involved in parasite structure, response against stress, chaperones, calcium-binding, and signal transduction were also identified. This work extends our knowledge of host-parasite relationships in the E. caproni-rodent model that is extensively used to analyze the factors determining the intestinal helminth rejection. Consequently, information on many proteins may be useful to better understand the molecular basis that determines the survival of this parasite in the definitive host.

Effects of Echinostoma caproni infection on the neutral and polar lipids of intestinal and non-intestinal organs in the BALB/c mouse as determined by high-performance thin-layer chromatography.

The goal of this study was to characterize and quantify the various neutral and polar lipid classes in the BALB/c mouse that are associated with Echinostoma caproni infection. Ten infected mice and 10 uninfected control mice were used for this study (five infected and five uninfected were used for each of the neutral lipid and polar lipid studies). After 3 weeks postinfection, the mice were necropsied and various organs were removed and prepared for lipid class analysis. The organs used were liver, kidney, spleen, colon, cecum, anterior portion of the small intestine (SI), middle portion of the SI, and posterior portion of the SI. Lipids were determined by high-performance thin-layer chromatography (HPTLC) with Analtech 10 x 20 cm HPTLC-HLF silica gel plates. For neutral lipids, petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase and 5% ethanolic phosphomolybdic acid detection reagent were used to determine the neutral lipids in each organ. Chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used to determine the polar lipids in each organ. The analyzed polar lipids in all organs were phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). Using HPTLC-densitometry for quantification, PC was found in the greatest amount and SM the smallest of all organs analyzed. The PE in the anterior portion of the SI was determined to be significantly greater (using the Student's t test with P < 0.05), with about twice the amount of PE in mice infected with E. caproni relative to the uninfected mice. No significant differences in any of the neutral lipid classes were found between infected and uninfected samples.

Echinostome-induced mortality varies across amphibian species in the field.

Echinostomes are receiving increased attention because of their emerging parasite status in landscapes associated with human development and their ability to infect and kill many North American larval amphibians. While laboratory experiments have shown that echinostomes can cause extensive mortality in their amphibian hosts, their effect on tadpoles in the field is less clear. I conducted a controlled-infection field-enclosure experiment in 4 ponds to compare the effects of echinostomes on green frog (Rana clamitans) and gray tree frog (Hyla versicolor) tadpoles in the field. I measured tadpole growth, development, mortality, and infection intensity. Echinostome infection resulted in high mortality in green frog tadpoles and less mortality in gray tree frogs. However, metacercariae encystment rates were higher in gray tree frog tadpoles than in green frog tadpoles. The effect of echinostomes on mortality varies across amphibian species, with the result that some species may experience more extensive echinostome-induced mortality than others. Mortality as a result of echinostome infection in green frog tadpoles was similar to mortality observed in predation experiments.

Physiological changes in Lymnaea columella (Say, 1817) (Mollusca, Gastropoda) in response to Echinostoma paraensei Lie and Basch, 1967 (Trematoda: Echinostomatidae) infection.

The physiological interaction between the digenean Echinostoma paraensei larvae and the intermediate snail host Lymnaea columella was studied. The carbohydrate content was significantly altered in the digestive gland tissue of snails, decreasing after 20 days postinfection. At the end of the prepatent period, the carbohydrate content was reduced by 60% when compared to uninfected snails. The total protein was reduced by 80.01% and 76.42% in the hemolymph and digestive gland, respectively. The end products resulting from nitrogen degradation were also analyzed. The urea was significantly increased in both the hemolymph and digestive gland. In the former, the highest concentration was detected at day 10 postinfection and then decreased until the end of the prepatent period. In digestive glands from infected snails, urea was significantly higher than in uninfected ones after 20 days. The uric acid content was reduced by 94.72% in the hemolymph and 43.75% in digestive glands after 10 days postinfection. The ammonia was undetectable under the experimental conditions employed.

The compatibiuty polymorphism in invertebrate host/trematodes interactions: research of molecular determinants.

The co-evolutionary dynamics that exist in many host-parasite interactions sometimes leads to compatibility polymorphism. This phenomenon is well documented in mollusc/trematodes interactions but its molecular base is unknown. In order to identify key molecules involved in this phenomenon, we developed several molecular approaches comparing compatible or incompatible strains of mollusc or parasite. These comparisons led to the identification of numerous candidate genes listed and discussed (some of them) in the present review.

High performance thin layer chromatographic analysis of neutral lipids in the urine of BALB/c mice infected with Echinostoma caproni.

Silica gel high performance thin layer chromatography-densitometry was used to determine the neutral lipid profile in the urine of BALB/c mice experimentally infected with adults of Echinostoma caproni. An approximate 30 worm burden per host induced certain changes in the neutral lipid profile of the mice at 2 to 10 weeks post-infection (PI) relative to the uninfected controls. Infection caused a significant increase (Student's t test, P < 0.05) in the methyl oleate fraction at 6 and 7 weeks PI relative to the uninfected controls. Neutral lipid profiles of urine may serve as an indicator of experimental infection of echinostomes in mice.

Development and pathology of Echinostoma caproni in experimentally infected mice.

In the present article, several parasitological features of mice, each experimentally infected with 75 metacercariae of Echinostoma caproni (Trematoda: Echinostomatidae), were studied during the first 12 wk postinfection. Moreover, the early pathological responses also were analyzed and compared with data previously published on other host species of E. caproni to gain further insight into the factors determining worm rejection or establishment of chronic infections. The results obtained show that the pattern of E. caproni infection in mice is consistent with a highly compatible host-parasite system. This combination is characterized by a high worm establishment, high egg output, and long survival of the worms. However, some differences with respect to other highly compatible hosts have been observed, particularly in relation to the survival of the adult worms. Histological studies suggest that the kinetics of goblet cells, mucosal neutrophils, and mononuclear inflammatory cells in the mesentery seem to be essential in determining the course of E. caproni infection in mice.

Effect of Echinostoma friedi (Trematoda: Echinostomatidae) experimental infection on longevity, growth and fecundity of juvenile Radix peregra (Gastropoda: Lymnaeidae) and Biomphalaria glabrata (Gastropoda: Planorbidae) snails.

The effect of Echinostoma friedi experimental infection on longevity, growth and fecundity of two susceptible first intermediate host snails, Radix peregra and Biomphalaria glabrata, was studied to contrast the level of compatibility. 120 R. peregra and 150 B. glabrata snails were used exposed to one, three or five miracidia and divided in three categories: INF (snails exposed and infected); ENI (exposed but not infected) and C (control or not miracidial-exposed snails). R. peregra INF snails' death process starts sooner, but in a prolonged extension, while B. glabrata INF snails have a much shorter life span. The infection and the miracidial exposure are able to reduce R. peregra normal development (stunting). B. glabrata INF snails' growth exceeds that of C snails (gigantism). E. friedi produces a total parasitic castration of R. peregra and B. glabrata INF snails. R. peregra would be considered as the required snail host, while B. glabrata only as an adequate snail host.

Immune response and inhibitory effect of ketotifen on the BALB/c and C3H/HeN mice infected with Echinostoma hortense.

Although Echinostoma hortense is one of the intestinal trematodes with a high infection rate in South Korea, the exact immune response against E. hortense infection has yet to be fully investigated. In the present study, we investigated differential susceptibilities in two different strains of micenamely, BALB/c (H-2d) and C3H/HeN (H-2k) mice. Likewise, we investigated the effects of ketotifen, an antiallergic drug, on the immune response against E. hortense infection. The worm recovery rate of the C3H/HeN mice was much higher than that of the BALB/c mice. The messenger ribonucleic acid (mRNA) expressions of interleukin (IL)-4 and IL-5 in the BALB/c mice were stronger than that of the C3H/HeN mice after E. hortense infection, but IL-1beta and tumor necrosis factor (TNF)-alpha expressions in the BALB/c mice were weaker than that of the C3H/HeN mice after E. hortense infection. The number of goblet cells and eosinophils increased after E. hortense infection in the BALB/c and the C3H/HeN mice. The worm recovery rate was higher and lasted longer in the ketotifen-treated mice in comparison to the untreated mice. Ketotifen suppressed the mRNA expression of IL-4 and IL-5 in the BALB/c mice, but did not in the C3H/HeN mice. The IL-1beta expressions were inhibited by ketotifen in the two strains, but TNF-alpha expression was inhibited in the C3H/HeN mice after ketotifen treatment. In addition, ketotifen inhibited the increase in eosinophils and goblet cells in varying degrees, depending on the strain. In summary, the immune sensitivity against E. hortense depends on the species of the host. The ketotifen treatment administered on the infected mice differently blocked the immune response against E. hortense infection.