Immune pathogenesis in pigeons during experimental Prohemistomum vivax infection
PMCID: PMC9516004
PMID:
Abstract
Prohemistomum vivax is a small trematode belonging to the family Cyathocotylidae, infecting fish-eating birds and mammals, including humans. However, no data on molecular identification and immune pathogenesis are available, challenging effective diagnostic and therapeutic interventions. Here, we identified P. vivax based on combined morphological and molecular data and examined histopathological lesions and the differential cytokines expression in experimentally infected pigeons. Pigeons were orally infected with 500 prohemistomid metacercariae. Intestinal and spleen tissues were harvested 2, 4, 7, 14, 21, and 28 days post-infection (dpi). Gene expression levels of eleven cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-γ, and TGF-β3) were assessed using quantitative reverse-transcription PCR (RT-qPCR). We identified the recovered flukes as Prohemistomum vivax based on morphological features and the sequence and phylogenetic analysis of the internal transcribed spacer 1 (ITS1), 5.8 ribosomal RNA, and ITS2 region. Histopathological lesions were induced as early as 2 dpi, with the intensity of villi atrophy and inflammatory cell infiltration increasing as the infection progressed. An early immunosuppressive state (2 and 4 dpi), with TGF-β3 overexpression, developed to allow parasite colonization. A mixed Th1/Th2 immune response (overexpressed IFN-γ, IL-12, IL-2, IL-4, and IL-5) was activated as the infection progressed from 7 to 28 dpi. Inflammatory cytokines (IL-1, IL-6, IL-18, and IL-15) were generally overexpressed at 7–28 dpi, peaking at 7 or 14 dpi. The upregulated Treg IL-10 expression peaking between 21 and 28 dpi might promote the Th1/Th2 balance and immune homeostasis to protect the host from excessive tissue pathology and inflammation. The intestine and spleen expressed a significantly different relative quantity of cytokines throughout the infection. To conclude, our results presented distinct cytokine alteration throughout P. vivax infection in pigeons, which may aid in understanding the immune pathogenesis and host defense mechanism against this infection.
Full Text
Cyathocotylidae Mühling, 1896 is a small, globally distributed digenean trematode family (superfamily Diplostomoidea Poirier, 1886), infecting birds, mammals, and reptiles (1). The taxonomy of this family has been controversial. According to most recent reports, this family is divided into five subfamilies, Cyathocotylinae Mühling, 1898; Prohemistominae Lutz, 1935; Szidatiinae Dubois, 1938; Prosostephaninae Szidat, 1936; and Muhlinginae Mehra, 1950. Prohemistomum vivax (P. vivax) belongs to the subfamily Prohemistominae which includes five genera: Prohemistomum Odhner, 1913; Mesostephanus Lutz, 1935; Mesostephanoides Dubois, 1951; Paracoenogonimus Katsurada, 1914; and Linstowiella Szidat, 1933 (1). Prohemistomum vivax inhabits the intestine of fish-eating birds and mammals, including humans, and has been recorded in Egypt, Israel (Palestine), Japan, and Europe (2, 3). This intestinal fluke can be transmitted by consuming infected fish intermediate hosts. Prohemistomum vivax intermediate hosts include a variety of fresh and brackish water fish, such as Tilapia zilli, Tilapia nilotica, Clarias gariepinus, Clarias lazera, Chrysichthys auratus, Bagrus bayad, Barbus binny, Ctenopharyngodon idella, Gambusia affinis, Shilbe mystus, Hydrocyon sp., Atherina sp., Alestes sp., Eutropius sp., Schilbe sp., Mugil capito, and Mugil cephalus (2, 4–7). Fish with metacercarial infection display myositis, muscle pressure atrophy, respiratory distress, excessive mucus, scale loss, and spots on affected tissues, with significant economic losses (7, 8). The zoonotic and economic impact of P. vivax necessitates developing an effective tool for diagnosis and control.
The current systematics of the family Cyathocotylidae mainly depend on morphological features. Molecular data and phylogenetic studies of this family are lacking. Globally, DNA sequences are available from adults of only four species from reptile hosts belonging to genera Suchocyathocotyle and Gogatea (9) and eight species from avian hosts belonging to genera Holostephanus, Holostephanoides, Cyathocotyle, Mesostephanus, and Neogogatea (9–13). Several cyathocotylid trematodes were recorded in Egypt, including Prohemistomum vivax, Prohemistomum azimi n. sp., Mesostephanus appendiculatus, Mesostephanus burmanicus, Mesostephanus milvi, Mesostephanus odhneri, and M. fajardensis (4, 14, 15). However, these species are morphologically similar, and molecular data were recovered from adults of only one species, M. appendiculatus (13). Thus, more efforts are needed to develop a fast and accurate molecular tool to differentiate these trematodes.
Intestinal trematode infections can cause significant pathological alterations in the gut of final hosts, leading to enteritis (16). Prohemistomum vivax in experimentally infected rats resulted in intestinal villi ulceration and deformation, shortening, blunting, and fusion. In the intestinal lamina propria, crypt hypertrophy with inflammatory cell infiltration was also observed (17). Although Amer (18) reported mast cells and eosinophils efflux in the intestinal mucosa of infected rats, intestinal mastocytosis plays a minor role in intestinal trematode expulsion (19). Therefore, more research is essential to clarify the immune mechanisms and effecter cells involved in host damage and worm expulsion during P. vivax infection.
In mammals, T-helper (Th) cell immunity, either Th1 or Th2, or both, is activated in response to helminth parasites. Th2 response is characterized by the production of interleukin (IL)-4, IL-5, IL-9, and IL-13. These interleukins trigger mast cells and eosinophils and raise IgE and IgG1 serum levels (20). Nevertheless, Th1 cells produce interferon (IFN)-γ and are linked to the release of proinflammatory cytokines, inhibiting Th2 responses. A successful resolution of infection requires balanced Th1 and Th2 responses, while an unbalanced response causes host damage (21). Like their mammalian counterparts, avian cytokines play a role in the host immune response to pathogenic infection. Ascaridia galli-infected pigeons had significantly higher IL1-β and tumor necrosis factor (TNF-α) levels than apparently healthy pigeons (22). IFN-γ and TNF-α cytokines were significantly upregulated in domestic pigeons during the late central-nervous phase of Apicomplexa protozoon parasite (Sarcocystis calchasi) infection (23). Nevertheless, little is known about cytokines' role during trematode infection in pigeons. Generally, the selective stimulation of Th1 and Th2 cells in helminthic infections differs depending on the parasite species. On the one hand, the host protection and worm expulsion during intestinal nematode infections mainly rely on the Th2 response (24). On the other hand, little is known about host Th responses in intestinal trematode infections, except for Echinostoma caproni and Neodiplostomum seoulense infections (25–27). Immune responses were biased toward a Th1 phenotype in E. caproni (25), while mixed Th1 and Th2 responses were activated during N. seoulense infection (26). However, no studies have investigated Th immune responses and related cytokines during infection with members of the Cyathocotylidae family. This study aims to identify P. vivax trematode based on morphology and internal transcribed spacer sequence and to evaluate the effect of this fluke on cytokines gene expression in the intestine and spleen of experimentally infected pigeons.
Encysted Metacercariae (EMC) were collected from infected African catfish (Clarias gariepinus), purchased from fish markets in Ismailia city, Ismailia Province, Egypt (30° 35′ 0″ N, 32° 16′ 0″ E). Minced muscle samples were exposed to the acid-pepsin solution (10 g 1:3000 pepsin powder (Oxford Lab Fine Chem LLP, Navghar, India), 10 mL of 25% HCl, and 2,000 mL distilled H2O; pH 2) and incubated at 37°C for 2–3 h with frequent stirring (28). After filtering the suspensions through a tea sieve, the filtrate was rinsed in 0.85% saline and examined under a stereomicroscope for metacercariae. The prohemistomid metacercariae were concentrated by sedimentation and morphologically identified according to Patarwut et al. (29). Metacercariae were counted and kept in 0.85% saline for <6 h before being used in the experimental infection on the same day.
Adult parasites were fixed in AFA (85 mL of 85% ethanol, 10 mL of formalin, and 5 mL of glacial acetic acid) for 10 min and then rinsed in 70% Ethanol for 3 min. Samples were stained in Semichon's acetocarmine stain for 5 min, washed twice in 70% Ethanol, de-stained in 1% acid alcohol for 5 s, and rinsed twice in alkaline alcohol for 3 min. Following dehydration in graded ethanol concentration, 95% (15 min) and 100% (twice, 15 min each), samples were cleared with xylene for 1 min and mounted on microscope slides in Canada balsam. Parasites were photographed using a Leica DM1000 microscope (Leica Microsystems, Wetzlar, Germany) at 100 × magnification, and measurements were obtained with the aid of the ImageJ software (LOCI, University of Wisconsin, USA). Our measurement data were presented as the range (mean ± SD). For taxonomic identification, we compared worm morphological features and measurement data to previous studies (3, 30–34).
Genomic DNA was extracted from 10 flukes stored at −20°C using a QIAGEN DNeasy™ tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The gDNA concentrations were evaluated by a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to optimize the amount of gDNA used in PCR reactions. The isolated gDNA was stored at −20°C until further use. To identify adult flukes, we selected the nuclear ribosomal internal transcribed spacer region, including part of internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA complete sequence, and part of ITS2. We used the universal primers BD1 (5′-GTCGTAACAAGGTTTCGGTA-3′) and BD2 (5′-TATGCTTAAATTCAGCGGGT-3′) (35, 36) synthesized by FASMAC Co. Ltd. (Atsugi, Japan). The PCR reactions (25 μL) comprised 12.5 μL EmeraldAmp MAX Master Mix (Takara Bio, Kusatsu, Japan), 1 μL of each primer (10 pmol/μL), 20 ng of template DNA, and ddH2O up to 25 μL. The PCR was run in a SensoQuest Labcycler 48 (SensoQuest GmbH company, Göttingen, Germany). PCR conditions were pre-denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 2 min; with a final extension at 72°C for 5 min. The amplified PCR products were examined by 1% ethidium bromide-stained agarose gel electrophoresis. After cleaning using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), PCR products were sent to Sangon Biotech Co., Ltd (Shanghai, China) for sequencing in both directions.
We determined homologous sequences using the Basic Local Alignment Search Tool (BLAST) on NCBI. Boundaries between ITS1, 5.8S rRNA, and ITS2 sequences were determined by aligning and comparing our sequence to that of Cyathocotyle prussica (MH521249). We selected 27 Diplostomata sequences for phylogenetic analysis. Selected fragments of the ITS1-5.8S-ITS2 region were assembled by MEGA X (37) and aligned with the Clustal W software (38). Homology percent and pairwise distances were analyzed using the Megalign module of the DNASTAR Lasergene package (v. 7.1.0). The alignments dataset was analyzed using MEGA X to predict the best-fitting nucleotide substitution model based on the Akaike Information Criterion (AIC). Maximum likelihood (ML) and Neighbor-joining (NJ) analyses were performed under the K2 + G model. Bootstrap values of 1,000 resampled datasets were used to estimate the nodal support of the phylogenetic tree. The tree explorer of MEGA X was utilized to visualize the phylogenetic trees. Clonorchis sinensis (MF319655) was used as an outgroup.
Small intestine tissue samples from each group were fixed in 10% formalin overnight, washed with ddH2O, dehydrated in ascending ethanol concentrations (30–100%), cleared with xylene, and embedded in paraffin wax. Sections of 3 μm thickness were sliced and dewaxed with xylene before rinsing in descending ethanol grades (100–30%) and ddH2O. After staining with hematoxylin and eosin (HE), sections were dehydrated. Slides were scanned, and images were processed using the ImageJ software (LOCI, University of Wisconsin, United States). Intestinal tissue sections were examined for pathological findings and scored for the following: in?ammation with villous atrophy (none = 0, slight = 1, moderate = 2, and severe = 3), in?amed area (mucosa = 1, mucosa and submucosa = 2, and transmural = 3), surface ulceration (none = 0, focal = 1, diffuse= 2, complete loss of surface epithelium = 3, entire surface epithelium and crypt epithelium are lost = 4), and involvement percentage (1–25% = 1, 26–50% = 2, 51–75% = 3, and 76–100% = 4) (39).
Gene expression of eleven cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-γ, and TGF-β3) in pigeon intestines and spleen at different time points were evaluated by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). We designed IL-2, IL-4, and IL-5 primers based on conserved regions in genes isolated from related birds available on NCBI. Primers for amplifying other cytokines and internal reference (β-actin) genes were synthesized based on published sequences (Table 1). The Applied Biosystems™ 7,500 Real-Time PCR Systems (Applied Biosystem, Bedford, MA, USA) was used to evaluate cytokine expression levels in pigeons during P. vivax infection. The qPCR system was consisted of 0.5 μL cDNA (750 ng), 0.5 μL of each forward and reverse primers (10 pmol/L), 5 μL WizPure™ qPCR Master (SYBR) (Wizbiosolution, Gyeonggi-do, Republic of Korea), 0.2 μL ROX Dye (50X), and ddH2O up to 10 μL. The qPCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min and 40 cycles of denaturation at 95°C for 15 s and annealing at 58°C for 1 min. The product specificity was detected by a melting curve program at 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Each experiment was conducted in triplicates. The relative expression levels of different cytokines were determined with β-actin as the reference gene using the 2−Δ method (40).
In preliminary experiments, high inoculation doses (1,000 and 2,000 metacercariae/pigeon) resulted in severe offensive greenish mucoid diarrhea 4 dpi. Pigeon mortality began at 5 dpi, and by 7 dpi, all birds died. However, 500 metacercariae per host infection did not affect pigeon survival, and pigeons only showed mild diarrhea and gradual weight loss. A 100% of challenged birds were infected. The eggs firstly appeared in feces on the fourth day post-infection. Eggs were large, oval-shaped, yellow, and measured 80–90 μm long by 50–62 μm wide (Figure 1A). On post-mortem examination, we mainly detected flukes in the upper intestine (duodenum and jejunum) of pigeons. Oral infection with 500 metacercariae resulted in the highest worm recovery rate (66.6 ± 5.2%) at 2 dpi, with an average of 333.3 ± 19.1 worms/ pigeon. The recovery rate gradually decreased as the infection progressed, with the lowest recovery rate (14.3 ± 1.7%, 71.7 ± 6.2 worm/ pigeon) observed in the 28-dpi group. Generally, there was a highly significant difference (p < 0.0001) in the worm recovery at different time points of infection. Multiple comparisons of the number of worms recovered per bird and recovery rate % at each time point showed a significant change between all groups, except for G2 vs. G3 and G4 vs. G5 comparisons (Table 2).
Based on morphological features, we identified the recovered flukes as Prohemistomum vivax (Sonsino, 1892). The body was undivided into two distinct regions, pyriform or oval, attenuated at both ends and wide at the middle part, concave ventrally. The oral sucker was round and subterminal, leading to a well-developed muscular pharynx. The ventral sucker was well-developed and spherical. The esophagus was short and bifurcated away from the ventral sucker into two intestinal caeca, terminating posterior to the posterior testes. Testes were tandem in position, the anterior one was smooth and ovoid, and the posterior one was quadrilateral. The cirrus pouch was well-developed and on the left side in the caudal region. The ovary was nearly pyramidal, situated laterally between two testes. Vitelline follicles were moderately large, confined in a horseshoe manner around the gonads, and postero-lateral to the tribocytic organ. The caudal appendage and vaginal sphincter were absent. The genital pore was subterminal (Figure 1B). Each organ of P. vivax adults was measured and compared to previous reports (Table 3). Statistical analyses of the measurement data showed significant differences in some parameters between this study and previous studies.
We determined the pairwise distance of the ITS1-5.8S-ITS2 region of P. vivax with those of 27 Diplostomata trematode sequences in GenBank (Figure 2). The Maximum Likelihood and Neighbor-joining phylogenetic analysis (Figure 3) had the same topology. Based on phylogenetic analysis and the pairwise distance comparison of our sample to the related sequences, our sample (P. vivax) belonged to a clade supported by 100% bootstrap, representing the trematode family Cyathocotylidae. Prohemistomum vivax was most closely related to cyathocotylid metacercariae isolated from common carp (Cyprinus carpio) fish from Hungary (MT668950) with an identity percent of 95.4%. Both samples clustered in one clade supported by a high bootstrap value (96/100%, ML/NJ). This clade clustered with that containing Mesostephanus sp. metacercariae isolates (HM064922-HM064924) from the pumpkinseed (Lepomis gibbosus) fish in Canada, with an identity percent (84.2–89%) to our sample.
Histopathological evaluation of intestinal sections from infected pigeons at different time points showed that P. vivax induced histopathological lesions as early as 2 dpi. The intensity of lesions progressively increased with the infection course (Figure 4, Table 4). Intestinal tissues from groups 1 to 6 revealed a variable degree of villi shortening and thickening or atrophy, and inflammatory cells infiltration in mucosal lamina propria. These alterations gradually accelerated as the infection progressed from 2 to 28 dpi, with the appearance of fused villi at 21 and 28 dpi (Figures 4A–F). Focal epithelial ulcerations were also detected in intestinal sections at time points following fluke maturity, in 7 and 14 dpi groups (Figures 4C,D). Flukes were seen between the intestinal villi and in the villi interspace without invading the crypt region. Some flukes were attached to the intestinal mucosa, pinching intestinal villi (Figure 4G). Intestinal tissues from uninfected pigeons (NC group) revealed normal long and slender uniform intestinal villi. Lamina propria occasionally showed few inflammatory cells with rounded uniform glands (Figure 4H). We used four parameters to evaluate the histopathological changes in pigeon intestines during infection (Table 4). The values of intestinal histological score gradually increased with the infection course, with the highest score (6) detected in 14-, 21-, and 28-dpi groups. The intensity of inflammatory infiltration and villous alterations gradually accelerated as the infection progressed from 2 to 28 dpi, with the induction of villous fusion at 21 and 28 dpi. Moreover, the involvement percentage increased from up to 25% in the 2-dpi group to 51–75% in the 21- and 28-dpi groups (Table 4).
To understand how the immune response altered throughout the infection, we compared the gene expression of eleven cytokines in the intestinal tissues of infected and uninfected pigeons using RT-qPCR. Results showed that P. vivax infection significantly increased Th1, Th2, Treg, and inflammatory cytokines gene expression (Figure 5, Table 5). Cytokines transcriptional trends revealed an immunosuppressive response during early infection stages, with decreased expression of some cytokines, such as IL-12, IL-4, IL-5, IL-1, IL-18, and IL-15, at 2 dpi, 4 dpi, or both compared to control. We did not detect any significant change in Th1 cytokines (IFN-γ, IL-12, and IL-2) gene expression during early infection. Increased IFN-γ mRNA signals were detected in small intestine 7 dpi (p = 0.0127) and peaked at 21 dpi (p < 0.0001). IL-12 mRNA signals increased in late infection during days 21–28 dpi. IL-2 significant expression levels were detected at 7–28 dpi, peaking at 7 dpi. IL-10 mRNA levels in the intestine showed a significantly higher expression than the control in late infection from 7 to 28 dpi, peaking at 28 dpi. TGF-β3 mRNA in intestine was rapidly elevated at 2 dpi (p = 0.0265), peaked at 4 dpi (p = 0.0135), remained significantly high until 7 dpi (p = 0.0418) followed by a non-significant change between 14 and 28 dpi compared to non-infected group. Fluke infection also upregulated Th2 cytokines (IL-4 and IL-5). IL-4 expression was significantly higher in infected groups than in the control group at 14–28 dpi, with the peak value recorded at 21 dpi (7.9-fold-change, p = 0.0044). IL-5 was significantly overexpressed at 7–28 dpi. IL-5 upregulation peaked at 7 dpi and then decreased toward late infection at 28 dpi, from 30 to 2.7 times the control. IL-1 inflammatory cytokine was significantly upregulated at 7 dpi (33-fold-change, p < 0.0001) in infected pigeons compared to control, remained high at 14 dpi (3.6-fold-change, p = 0.0101), and then decreased to basal level. A significantly higher expression level of IL-18 was only detected at 7 dpi (35-fold-change, p = 0.0064). IL-6 transcription level significantly increased in the period from 4 to 28 dpi, with the highest level recorded at 7 dpi (12.8-fold-change, p = 0.0015). IL-15 upregulation was observed at 7–28 dpi, with a gradual decrease toward the end of the experiment.
Figure 6 and Table 6 show changes in cytokine mRNA expressions in the spleen of P. vivax-infected pigeons. Increased IFN-γ mRNA signals were detected at 7–28 dpi, peaking at 21 dpi (29-fold-change, p = 0.0010). IL-12 mRNA signals significantly increased during days 14–28 PI, with the highest value recorded at 14 dpi and the lowest detected at 28 dpi. IL-2 cytokine expression significantly increased at 7–28 dpi. A significant increase in IL-10 mRNA levels was detected late at 14 dpi, with the peak (10.8 times that of the control group) occurring at 21 dpi (p = 0.0036). TGF-β3 mRNA was significantly elevated throughout the infection from 2 to 28 dpi. However, the fold change of upregulation peaked at 2 dpi (31.9-fold-change, p < 0.0001) and gradually decreased as the infection progressed. Th2 cytokines expression significantly increased at 7–28 dpi for IL-5 and 14–28 dpi for IL-4. The peak expression level was detected at 21 dpi for IL-4 and IL-5. IL-1 cytokine was significantly upregulated at 14–28. Significantly higher expression levels of IL-6 and IL-18 were found at 7–28 dpi. IL-15 upregulation was observed at 4–28 dpi.
Pearson's correlation analysis was conducted on each pair of cytokines based on relative expression quantity (RQ) from infected groups (Table 7). In the intestine, this analysis showed a significant positive association between IFN-γ mRNA expression and IL-2 (r = 0.86, p < 0.0001) and Th2 cytokine (IL-4: r = 0.61, p = 0.0072) as well as between IL-2 and IL-5 (r = 0.64, p = 0.0042). IL-10 gene expression was significantly associated with both Th1 (IFN-γ: r = 0.48, p = 0.0461; IL-12: r = 0.92, p < 0.0001) and Th2 cytokines (IL-4: r = 0.49, p = 0.0380). TGF-β3 relative mRNA expression demonstrated a significant negative correlation to the other cytokines (IFN-γ: r = −0.65, p = 0.0032; IL-12: r = −0.76, p = 0.0002; IL-4: r = −0.70, p = 0.0011; IL-10: r = −0.79, p < 0.0001).
In spleens, a strong positive correlation was also detected between IFN-γ and Th2 cytokine expressions (IL-4: r = 0.86, p < 0.0001; IL-5: r = 0.82, p < 0.0001). IL-12 mRNA was positively correlated to Th2 cytokines (IL-4: r = 0.66, p = 0.003; IL-5: r = 0.51, p = 0.0315) and other Th1 cytokines (IFN-γ: r = 0.61, p = 0.0068; IL-2: r = 0.50, p = 0.0352). A strong positive correlation was detected between IL-4 and IL-5 (r = 0.94, p < 0.0001). IL-10 expression in spleen was positively correlated with both Th1 and Th2 cytokines (IFN-γ: r = 0.76, p = 0.0002; IL-12: r = 0.66, p = 0.0030; IL-4: r = 0.91, p < 0.0001; IL-5: r = 0.83, p < 0.0001). The relative mRNA expression of TGF-β3 was negatively correlated to other cytokines (IFN-γ: r = −0.83, p < 0.0001; IL-12: r = −0.62, p = 0.0056; IL-4: r = −0.84, p < 0.0001; IL-5: r = −0.68, p = 0.0021; IL-10: r = −0.86, p < 0.0001), in Table 7.
Table 8 compares relative cytokine expressions between the intestine and spleen of P. vivax-infected pigeons. Our findings revealed a significant difference in the relative quantity of cytokines between the two organs throughout the infection. Th1 (IFN-γ and IL-12), IL-4, TGF-β3, and IL-15 cytokines had significantly higher expression in the spleen than in the intestine. However, at 7, 21, and 28 dpi, IL-2 and IL-10 levels in the intestine were significantly higher than those in the spleen. Other cytokines (IL-5, IL-1, IL-6, and IL-18) were significantly higher in the intestine at early infection but became higher in the spleen as the infection progressed. IL-5 levels were higher in the intestine than in the spleen at 2–14 dpi, with a significant difference at 7–14 dpi. Then, at 21–28 dpi, IL-5 levels in the spleen were higher than in the intestine. The intestine had significantly higher expression levels of IL-1 at 7 dpi, IL-6 at 4–7 dpi, and IL-18 at 2 and 7 dpi, while the spleen had higher levels of all three cytokines at 14–28 dpi.
In this study, the morbidity and fatality in infected pigeons depended on the infection dose. The ingestion of 1,000 or more metacercariae resulted in the death of all birds within a week of infection, while 500 metacercariae inoculum did not cause fatalities in pigeons during the studied period of 28 days. Similarly, the severity of the clinical signs and the mortality in Neodiplostomum seoulense-experimentally infected mice was proportional to the metacercariae inoculum. Mice receiving 1,000 N. seoulense metacercariae died within 16 days post-infection. The possible causes of death might be excessive fluid loss and malnutrition due to malabsorption, mucosal bleeding, and persistent diarrhea (2). The earlier death in our study compared to that recorded with N. seoulense might be related to the difference in host and parasite species. We showed that metacercariae could successfully develop into adult P. vivax in the intestine of pigeons. This result agreed with previous studies (5, 7). P. vivax characteristic eggs were firstly detected in pigeon feces 4 days post-infection, in agreement with previous results in mice and rats (17, 34). Mahfouz et al. (17) suggested that the parasite's early growth may necessitate a rich supply of nutrients. Thus, it is possible to detect the parasite engulfing the epithelial villi. The upper intestine was the primary habitat of P. vivax in the final host (pigeons), consistent with results reported in P. vivax-infected mice (17, 18).
The recovered flukes were confirmed as P. vivax based on combined morphological and molecular identification. Although, there was a significant difference between our sample measurements and that of previous studies in some parameters, our sample matched all morphological characters of P. vivax reported in previous studies (3, 30, 32–34). Our sample was smaller than that recorded by Fahmy and Selim (32) and El-Nafffar et al. (33). This difference might be related to the different biological environments in the intestine of variable hosts (animals and birds) (42) and the difference between experimental and natural infection. For example, we compared measurements of P. vivax recovered from human (3), naturally infected dogs (32), experimentally infected dogs and cats (33), and experimentally infected rats (34) Similarly, Fahmy et al. (42) showed that P. vivax recovered from dogs were larger than that collected from buff-backed herons. We successfully amplified the ITS1, 5.8 S rRNA, and ITS2 region from P. vivax for the first time. In the phylogenetic tree, our sample (P. vivax) belonged to the family Cyathocotylidae clade. P. vivax was closely related to cyathocotylid metacercariae recovered from Hungarian common carp fish (95.4% homology) and clustered in one clade with Mesostephanus sp. metacercariae, which might represent the subfamily prohemistominae clade. These findings agreed with our sample morphological description and known taxonomy.
In histopathological examination, we found P. vivax flukes between the intestinal villi and never invaded the crypt region, similar to findings in mice (17). P. vivax caused histopathological alterations as early as 2 dpi. Furthermore, lesion intensity and intestinal histological score gradually increased with the infection course, with the highest score (6) detected on 14, 21, and 28 dpi. Intestinal tissue lesions included shortening and thickening or atrophy of villi, villous fusion, inflammatory cell infiltration in mucosal lamina propria, and focal epithelial ulcerations. Similar pathological changes were seen in the upper intestine of P. vivax-infected pigeons and mice (7, 17), Heterophyes heterophyes-infected mice (43), Echinostoma malayanum-infected hamsters, rats, mice, and gerbils (44), Metagonimus yokogawai-infected cats, N. seoulense-infected rats and mice, Pygidiopsis summa-infected rats and mice, and Gymnophalloides seoi-infected mice, reviewed by Chai (2). The observed pathological alterations might be attributed to the parasite's direct effect and the host's response. Intestinal flukes, including P. vivax, can induce mechanical and chemical damage in the host gut. P. vivax possesses a spiny tegument and well-developed adhesive tribocytic organ (45), which may directly affect parasite pathogenesis due to irritation of the host mucosa (17). Additionally, the tribocytic organ of diplostomidean flukes can pierce the host villi and secrete alkaline phosphatase that can lyse the villi (2). Early studies proposed that frequent movement and rotation of the trematode anterior part injury the intestinal mucosa and result in mechanical pressure on adjacent villi (46). Chai (47) suggested that the villi pressure atrophy in M. yokogawai-infected rat intestine might be related to the accumulation of mucus and gases in the gut lumen.
Previous reports have supported the induction of host protection mechanisms against P. vivax, activating the humoral and cellular host immune responses (17). Helal et al. (48) observed a massive influx of eosinophils and mast cells in the intestines of infected rats. Still, no studies have investigated the molecular pathology and the immune effector mechanism of intestinal infection with P. vivax. The immune response is regulated by CD4 T cells that are primarily classified into Th1 or Th2 T cells based on the released cytokines. Th1 cells mainly release IFN-γ and IL-12, while Th2 cells release IL-4, IL-5, IL-9, IL-10, and IL-13 (49, 50). These cell subsets induce different and counterregulatory immune responses. Th2 response cytokines regulate the host protection, whereas Th1 responses are related to chronic infections (25). Nevertheless, the preferential stimulation of Th1 and Th2 cells during parasite infections depends on many variables, including host genetic nature, parasite species, infection route and stage, and acute or chronic infection (25, 26).
This study demonstrated that P. vivax infection significantly impacted Th1, Th2, Treg, and inflammatory cytokines gene expression. The cytokine transcriptional profile indicated an immunosuppressive response in the early stages, a mixed Th1/Th2 response as the infection progressed, and a late tilting toward Th1/Treg response. At early infection (2–4 dpi), the expression of some cytokines was generally lower than in controls, implying an immunosuppressive condition to facilitate parasite colonization. This early immunosuppression state and cytokine decline were also observed during Fasciola gigantica infection and were attributed to the overexpression of the immunosuppressive cytokine TGF-β at 3 dpi (50). In agreement with this hypothesis, our study showed that TGF-β3 mRNA was rapidly elevated in the intestine at 2 dpi and remained significantly high until 7 dpi, followed by a non-significant change at 14–28 dpi. Although TGF-β3 mRNA in the spleen was significantly high throughout the infection period, the fold change of expression peaked at 2 dpi and gradually diminished as the infection progressed. These results suggest that TGF-β3 might play a role in immunosuppression and parasite colonization during early P. vivax infection. Similarly, semi-quantitative RT-PCR revealed an upregulation in TGF-β expression in P. vivax-infected mice intestines during the examined first 6 days of infection (17). TGF-β was also overexpressed during various parasitic infections, such as Schistosoma japonicum (51), Echinococcus granulosus (52), and Heligmosomoides polygyrus (53). We detected a significant negative correlation between TGF-β3 and other cytokines in both intestines and spleen of infected groups. Although there was no significant difference, TGF-β was also inversely related to other cytokines during F. gigantica infection (50). This negative correlation might be related to the immunosuppressive role of TGF-β. TGF-β can suppress T-cell proliferation, cytokine production and cytotoxicity, IFN-γ production, MHC class II expression, and reactive oxygen intermediates and nitric oxide release from activated macrophages (54).
Here, as P. vivax infection progressed 7–28 dpi, a mixed Th1 and Th2 responses developed in agreement with results of Shin et al. (26), who showed that mixed Th1 and Th2 responses shared in the activation of humoral and cellular immune reactions throughout N. seoulense infection in mice. P. vivax infection upregulated Th1 (IFN-γ, IL-12, and IL-2) and Th2 cytokines (IL-4 and IL-5). IFN-γ is released from Th1 cells and is linked to proinflammatory cytokines production, inhibiting Th2 responses (26). IL-12 is critical for enhancing cell-mediated immune responses. IL-12 and IL-12R-deficient humans and mice had impaired immune responses and were vulnerable to intracellular pathogen infections. IL-12 plays an important role in Th cell differentiation into the Th1 subset. IL-12 also stimulates IFN-γ release, in synergy with IL-2 or IL-18, and T and NK cell proliferation and cytolytic activity (55). IL-2 was the first cytokine to be molecularly cloned and was identified as a T cell growth factor vital for T cell proliferation and effector and memory cell generation. Subsequent research showed that IL-2 is necessary for maintaining Treg cells, and its absence results in an intense Treg cell deficiency and autoimmunity because IL-2 encourages Treg cell generation, survival, and activity. Therefore, IL-2 serves two contradictory roles: enhancing conventional T cells to boost immune responses and maintaining Treg cells to control immune responses (56). We found that IFN-γ was upregulated in both the intestine and the spleen (at 7 dpi) before IL-12, which may be contradictory. Overexpression of IFN-γ also proceeded IL-12 upregulation in the spleen and mesenteric lymph node of N. seoulense-infected mice, but not in the small intestine (26). Although IL-12 is a major inducer of IFN-γ, IL-2 also significantly impacts IFN- expression. These cytokines (IL-12 and IL-2) can induce IFN-γ expression independently. However, they act synergistically to induce large amounts of IFN-γ, particularly from NK cells (57). At 7 dpi, we found IL-2 upregulation in the intestine and spleen, with 29- and 17-fold increases compared to controls, respectively. The overexpressed IL-2 might have induced IFN-γ production either independently or in synergy with the low amount of IL-12 expressed during early infection. IFN-γ and IL-12 expression is coordinated, meaning that IL-12 induces IFN-γ, and IFN-γ induces IL-12 (58). Therefore, when the expression of IFN-γ increased, IL-12 was upregulated, which in turn exaggerated IFN-γ release. IL-4 is produced by Th2 cells and is essential for Th2 cell differentiation, as well as B cell activation, enabling IgE production. This cytokine plays a crucial role in parasite infections, promoting the host's resistance to parasite invasion (59). IL-5 is a cytokine that is involved in the Th2-type inflammatory response in the host and triggers eosinophil production and stimulation (60). In this study, Th1 and Th2 cytokine expressions were positively correlated, consistent with the findings of Shi et al. (50). This positive correlation might be related to the antagonistic functions of both responses and could be essential for immune homeostasis.
Intestinal nematodes, such as Heligmosomoides polygyrus, Trichuris muris, and Nippostrongylus brasiliensis, can induce strong Th2 responses (20, 24). On the contrary, intestinal trematodes, such as N. seoulense, can activate mixed Th1 and Th2 responses, but Th1 cytokine production (IFN-γ and IL-12) was the more potent among them (26). This result agreed with observations for Echinostoma caproni (25), which mainly triggered Th1 responses (elevated IFN-γ and IL-12) while also increasing Th2 cytokines (IL-5 and to a lower extent, IL-4). A single injection of anti-IFN-γ antibodies to E. caproni-infected mice significantly reduced worm burden, suggesting the IFN-γ role in establishing chronic echinostomiasis (25). Host fatalities were recorded in N. seoulense and P. vivax but not E. caproni infections. Thus, chronic infection is linked to a Th1 response (25), while mixed Th1 and Th2 responses may contribute to immunopathogenesis that results in host injury and worm expulsion (26). In N. seoulense-infected mice spleens and intestines, numbers of macrophages, activated either by Th1 or Th2 cells, were increased at 14–28 dpi (26), the expected period for worm expulsion (46). In vitro, rat macrophages eliminated N. seoulense through antibody-dependent cellular cytotoxicity. As a result, Shin et al. (26) concluded that combined Th1 and Th2 responses could result in extensive macrophage infiltration in the infected rat spleen and small intestine, enhancing worm ejection and host damage. Although this conclusion might be functional during the infection with P. vivax, a diplostomidean fluke related to N. seoulense, more studies are required to assess the role of macrophages in P. vivax.
Treg cytokine IL-10 mRNA levels were significantly upregulated at 7–28 dpi in the intestine and 14–28 dpi in the spleen. This result agreed with IL-10 upregulation in other helminthic infections, such as N. seoulense (26), E. caproni (27), Fasciola gigantica (50), Toxocara canis (61), Ostertagia ostertagi (62), Strongyloides venezuelensis (63), and Trichinella spiralis (64). IL-10 is an anti-inflammatory cytokine released from Treg cells that can reduce the immunopathological damage in the host by suppressing parasite-induced inflammation (65). IL-10 is also critical for Th2 response and induces T cell response shifting to the Th2 type while acting as a suppressive regulator of IL-12 (Th1) and inhibiting Th1 cell differentiation (65, 66). We suggest that IL-10 expression increased during P. vivax infection to counteract the high levels of inflammatory cytokines and maintain the Th1/Th2 balance and immune homeostasis, thereby protecting the host from excessive inflammation and tissue pathology. In our study, there was a positive correlation between IL-10 and Th1 and Th2 cytokines, consistent with the results in F. gigantica-buffaloes recorded by Shi et al. (50). Increasing IL-10 levels with the concurrent upregulation of Th1 and Th2 cytokines suggest that IL-10 is required for the Th2 response to P. vivax infection. Moreover, IL-10 overexpression follows Th1 response rise to hinder the overexaggerated Th1 cell activity, maintaining Th1/Th2 balance and immune homeostasis.
IL-1 inflammatory cytokine was significantly upregulated at 7–14 dpi in the intestine and 14–28 dpi in the spleen. Similarly, the cytokine IL-1β mRNA was overexpressed in the livers of F. gigantica-infected buffaloes (50). We also detected a significantly higher IL-18 expression level at 7 dpi in the intestine and 7–28 dpi in the spleen, in agreement with Alhallaf et al. (67), who showed elevated IL-18 secretion in humans and mice infected with Trichuris nematodes. The IL-1 family of cytokines (IL-1α, IL-1β, and IL-18) is released from innate immune cells, such as macrophages, monocytes, dendritic cells, endothelial, and epithelial cells, and are essential for initiating and enhancing innate and adaptive immune responses, resisting microbial infections, regulating immune response (67, 68). IL-18 can promote Th2-type immune responses via stimulating IL-4 and IL-13 production from basophils in the presence of a second stimulus (IL-3). Additionally, IL-18 plays a vital role in priming NK cells for IFN-γ production and increases its secretion in response to IL-12 (68). IL-1α and IL-1β have many immunomodulatory roles, including activating monocytes and macrophages, stimulating B cell proliferation and differentiation in collaboration with IL-4 and IL-6, and potentiating antibody production (69). Some studies suggest that IL-1α and IL-1β have a physiological impact on Th cell response development. IL-1 can enhance T cell proliferation by stimulating the transcription of IL-2 receptor (70). Moreover, lymph node cells from IL-1α and β-deficient mice failed to differentiate into Th2 cells in the presence of IL-4. Thus, IL-1α and IL-1β are required, along with IL-4, for Th2 response development. In vivo, IL-1α and IL-1β were involved in establishing Th2-mediated resistance (expulsion) to nematodes, where IL-1α and IL-1β-deficient mice were vulnerable to chronic Trichuris muris infection, with impaired Th2 responses (69). IL-1 has also been essential for IL-12-induced Th1 cell growth (71). Concurrent overexpression of IL-1 and IL-18 with Th1 and Th2 cytokines in P. vivax infection might support previous suggestions for their role in developing Th1 and Th2 responses.
We demonstrated that IL-6 transcription levels significantly increased in the intestine at 4-28 dpi and in the spleen at 7-28 dpi. Similar to our results, E. caproni-infected rats and mice showed a significant increase in IL-6 in the spleen and intestine (27). IL-6 activates T helper cells, regulates T cell resistance to apoptosis, and modulates the Treg/Th17 cells balance (72). IL-6 acts as a myokine, i.e., a cytokine released from muscle during contraction (27, 73). IL-6 expression was more marked in the intestine of E. caproni-infected rats (resistant host) compared to mice (compatible host), suggesting the possible role of IL-6 in intestinal motility in response to intestinal trematode (27). IL-6 upregulation might alter intestinal smooth muscle motility, hindering worm attachment and feeding in the intestine, thus helping worm expulsion during P. vivax infection. Additionally, the high expression of Th1 cytokines (IL-1, IL-18, and IL-6), indicating acute inflammatory response, might be linked to the severe inflammatory cell infiltrations observed in the pigeon intestine.
We observed IL-15 upregulation at 7–28 dpi in P. vivax-infected pigeons' intestines and 4–28 dpi in spleens. IL-15 is a proinflammatory cytokine involved in the development, proliferation, and activation of several lymphocyte lineages (74). This cytokine acts as a pleiotropic lymphokine that is not expressed in T cells but is derived from many other cells and tissues, including skeletal tissue, muscle, placenta, lung, kidney, heart, epithelial cells, fibroblasts, and monocytes (75). IL-15 belongs to the common cytokine receptor common gamma chain (γc) family of cytokines (with IL-2, IL-4, IL-7, IL-9, and IL-21), playing essential roles in innate and adaptive immunity. IL-15 receptor (IL-15R) shares the β chain and γ chain of IL-2R. Therefore, IL-15 shares some immunological activities with IL-2, such as stimulating CD4+ and CD8+ T-cell proliferation and activation, T helper cells differentiation, B cells antibodies synthesis, natural killer (NK) cell proliferation and maintenance, and CD8+T cells and NK cells cytolytic activity (76). However, IL-15 activities on T cell-mediated immune responses are distinct from those of IL-2 because IL-15 uses a different α-chain from IL-2R and expresses a specific receptor (IL-15RX). IL-15 can suppress the IL-2-mediated T cell responses and Fas-mediated activation-induced cell death (AICD) process. In contrast to IL-2, IL-15 has little effect on Treg activity, but it can compromise Treg function by acting on CD4+T and CD8+T cells (75). IL-15 can boost both Th1 responses by enhancing IFN-γ and TNF-α production and Th2 responses by enhancing IL-4 and IL-5 release (75, 77). Studies have revealed that IL-15 might play a role in resisting intracellular protozoan infections, including Leishmania braziliensis, Cryptosporidium, Plasmodium, and Toxoplasma gondii, by influencing Th1 or Th2 response development (77–81). Although little is known about the role of IL-15 in helminth infections, some studies indicated that IL-15 plays a role in the immune response against helminth parasites similar to that played in protozoan parasite infections. The mRNA levels of IL-15 in the immunized group slightly exceeded those found in the O. ostertagi-infected group (82). Filarial nematode (Setaria equina) excretory-secretory antigens stimulated IL-15 production in mice (83).
The immune response was quite different between the intestine and spleen tissues of P. vivax-infected pigeons. The Th1 response (IFN-γ and IL-12) in the intestines appeared more persistent until the end of the experiment, while Th2 cytokine (IL-5) expression fold change peaked at 7 dpi and then decreased toward late infection at 28 dpi. However, the general trend of both Th1 and Th2 cytokines in the spleen was nearly similar, significantly overexpressed at 7–28 or 14–28 dpi and peaking at 14 or 21 dpi. Similarly, Trelis et al. (27) recorded a marked Th1 response (IFN-γ) in the intestine and a mixed Th1/Th2 phenotype in the spleen of E. caproni-infected high compatible host (mice).
On comparing the relative cytokine quantity in the intestine and spleen, we observed a significant difference in the cytokine expression levels between the intestine and the spleen throughout the infection. Intestinal epithelial cells represent the first barrier of enteric immunity. Helminths cause intestinal tissue damage during feeding, stimulating the release of cytokine alarmins, such as IL-25, IL-33, and thymic stromal lymphopoietin. These alarmins triggers innate lymphoid 2 cells, a major source of the Th2 cytokines (IL-5 and IL-13). As a result, IL-5 and IL-13 levels can rise within hours of helminth infection (84). These findings could explain the early higher expression of IL-5 in the intestine than in the spleen. The lamina propria provides an influential immune cell population, quickly activated after intestinal helminth infection (84). Inflammatory cell infiltration in the lamina propria was detected in the intestine of P. vivax-infected pigeons as early as 2 dpi, which could be related to the higher expression of inflammatory cytokines (IL-1, IL-6, and IL-18) in the intestine during early infection. Additionally, helminth excretory/secretory products and activated dendritic cells circulate to several organs, including the spleen, where they contribute to systemic response development. The spleen is a major source of serum immunoglobulins and Th2 cells. Two weeks after infection with H. polygyrus, splenomegaly was observed, with augmented type 2 cytokine expression. Thus, the intestinal mucosal response develops first and is accompanied by higher Th2 cytokine levels, whereas the systemic response (spleen) quickly follows the initial enteric response (84). This hypothesis could explain IL-5, IL-1, IL-6, and IL-18 increased expression in the spleen after 2 weeks of infection following the upregulation in the intestine. Although the spleen expressed more IFN-γ, IL-12, IL-4, TGF-3β, and IL-15 than the intestine, the difference was mainly significant after 7 dpi, possibly after the systemic response initiation. Even though not statistically analyzed, the spleen of N. seoulense-infected mice had a higher relative quantity of IFN-γ and IL-4 than the intestine (26), similar to our results. The type 2 immune response at the site of intestinal helminth infection maintains the stimulates of Th2 effector cells and T regulatory cells, which may lead to higher IL-10 levels in the intestine than in the spleen. This upregulation was maintained in the intestine till 28 dpi, perhaps to hinder the increased inflammatory cytokine expressions in the intestine and protect the host from pathological damage. Like IL-10, IL-2 was highly expressed in the intestine compared to the spleen, which might be related to IL-2 role in maintaining Treg cell survival and activity.
Sections
"[{\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B1\", \"B1\", \"B2\", \"B3\", \"B2\", \"B4\", \"B7\", \"B7\", \"B8\"], \"section\": \"Introduction\", \"text\": \"Cyathocotylidae M\\u00fchling, 1896 is a small, globally distributed digenean trematode family (superfamily Diplostomoidea Poirier, 1886), infecting birds, mammals, and reptiles (1). The taxonomy of this family has been controversial. According to most recent reports, this family is divided into five subfamilies, Cyathocotylinae M\\u00fchling, 1898; Prohemistominae Lutz, 1935; Szidatiinae Dubois, 1938; Prosostephaninae Szidat, 1936; and Muhlinginae Mehra, 1950. Prohemistomum vivax (P. vivax) belongs to the subfamily Prohemistominae which includes five genera: Prohemistomum Odhner, 1913; Mesostephanus Lutz, 1935; Mesostephanoides Dubois, 1951; Paracoenogonimus Katsurada, 1914; and Linstowiella Szidat, 1933 (1). Prohemistomum vivax inhabits the intestine of fish-eating birds and mammals, including humans, and has been recorded in Egypt, Israel (Palestine), Japan, and Europe (2, 3). This intestinal fluke can be transmitted by consuming infected fish intermediate hosts. Prohemistomum vivax intermediate hosts include a variety of fresh and brackish water fish, such as Tilapia zilli, Tilapia nilotica, Clarias gariepinus, Clarias lazera, Chrysichthys auratus, Bagrus bayad, Barbus binny, Ctenopharyngodon idella, Gambusia affinis, Shilbe mystus, Hydrocyon sp., Atherina sp., Alestes sp., Eutropius sp., Schilbe sp., Mugil capito, and Mugil cephalus (2, 4\\u20137). Fish with metacercarial infection display myositis, muscle pressure atrophy, respiratory distress, excessive mucus, scale loss, and spots on affected tissues, with significant economic losses (7, 8). The zoonotic and economic impact of P. vivax necessitates developing an effective tool for diagnosis and control.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B9\", \"B9\", \"B13\", \"B4\", \"B14\", \"B15\", \"B13\"], \"section\": \"Introduction\", \"text\": \"The current systematics of the family Cyathocotylidae mainly depend on morphological features. Molecular data and phylogenetic studies of this family are lacking. Globally, DNA sequences are available from adults of only four species from reptile hosts belonging to genera Suchocyathocotyle and Gogatea (9) and eight species from avian hosts belonging to genera Holostephanus, Holostephanoides, Cyathocotyle, Mesostephanus, and Neogogatea (9\\u201313). Several cyathocotylid trematodes were recorded in Egypt, including Prohemistomum vivax, Prohemistomum azimi n. sp., Mesostephanus appendiculatus, Mesostephanus burmanicus, Mesostephanus milvi, Mesostephanus odhneri, and M. fajardensis (4, 14, 15). However, these species are morphologically similar, and molecular data were recovered from adults of only one species, M. appendiculatus (13). Thus, more efforts are needed to develop a fast and accurate molecular tool to differentiate these trematodes.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B16\", \"B17\", \"B18\", \"B19\"], \"section\": \"Introduction\", \"text\": \"Intestinal trematode infections can cause significant pathological alterations in the gut of final hosts, leading to enteritis (16). Prohemistomum vivax in experimentally infected rats resulted in intestinal villi ulceration and deformation, shortening, blunting, and fusion. In the intestinal lamina propria, crypt hypertrophy with inflammatory cell infiltration was also observed (17). Although Amer (18) reported mast cells and eosinophils efflux in the intestinal mucosa of infected rats, intestinal mastocytosis plays a minor role in intestinal trematode expulsion (19). Therefore, more research is essential to clarify the immune mechanisms and effecter cells involved in host damage and worm expulsion during P. vivax infection.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B20\", \"B21\", \"B22\", \"B23\", \"B24\", \"B25\", \"B27\", \"B25\", \"B26\"], \"section\": \"Introduction\", \"text\": \"In mammals, T-helper (Th) cell immunity, either Th1 or Th2, or both, is activated in response to helminth parasites. Th2 response is characterized by the production of interleukin (IL)-4, IL-5, IL-9, and IL-13. These interleukins trigger mast cells and eosinophils and raise IgE and IgG1 serum levels (20). Nevertheless, Th1 cells produce interferon (IFN)-\\u03b3 and are linked to the release of proinflammatory cytokines, inhibiting Th2 responses. A successful resolution of infection requires balanced Th1 and Th2 responses, while an unbalanced response causes host damage (21). Like their mammalian counterparts, avian cytokines play a role in the host immune response to pathogenic infection. Ascaridia galli-infected pigeons had significantly higher IL1-\\u03b2 and tumor necrosis factor (TNF-\\u03b1) levels than apparently healthy pigeons (22). IFN-\\u03b3 and TNF-\\u03b1 cytokines were significantly upregulated in domestic pigeons during the late central-nervous phase of Apicomplexa protozoon parasite (Sarcocystis calchasi) infection (23). Nevertheless, little is known about cytokines' role during trematode infection in pigeons. Generally, the selective stimulation of Th1 and Th2 cells in helminthic infections differs depending on the parasite species. On the one hand, the host protection and worm expulsion during intestinal nematode infections mainly rely on the Th2 response (24). On the other hand, little is known about host Th responses in intestinal trematode infections, except for Echinostoma caproni and Neodiplostomum seoulense infections (25\\u201327). Immune responses were biased toward a Th1 phenotype in E. caproni (25), while mixed Th1 and Th2 responses were activated during N. seoulense infection (26). However, no studies have investigated Th immune responses and related cytokines during infection with members of the Cyathocotylidae family. This study aims to identify P. vivax trematode based on morphology and internal transcribed spacer sequence and to evaluate the effect of this fluke on cytokines gene expression in the intestine and spleen of experimentally infected pigeons.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B28\", \"B29\"], \"section\": \"Parasite isolation\", \"text\": \"Encysted Metacercariae (EMC) were collected from infected African catfish (Clarias gariepinus), purchased from fish markets in Ismailia city, Ismailia Province, Egypt (30\\u00b0 35\\u2032 0\\u2033 N, 32\\u00b0 16\\u2032 0\\u2033 E). Minced muscle samples were exposed to the acid-pepsin solution (10 g 1:3000 pepsin powder (Oxford Lab Fine Chem LLP, Navghar, India), 10 mL of 25% HCl, and 2,000 mL distilled H2O; pH 2) and incubated at 37\\u00b0C for 2\\u20133 h with frequent stirring (28). After filtering the suspensions through a tea sieve, the filtrate was rinsed in 0.85% saline and examined under a stereomicroscope for metacercariae. The prohemistomid metacercariae were concentrated by sedimentation and morphologically identified according to Patarwut et al. (29). Metacercariae were counted and kept in 0.85% saline for <6 h before being used in the experimental infection on the same day.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B3\", \"B30\", \"B34\"], \"section\": \"Morphometric analysis of parasites\", \"text\": \"Adult parasites were fixed in AFA (85 mL of 85% ethanol, 10 mL of formalin, and 5 mL of glacial acetic acid) for 10 min and then rinsed in 70% Ethanol for 3 min. Samples were stained in Semichon's acetocarmine stain for 5 min, washed twice in 70% Ethanol, de-stained in 1% acid alcohol for 5 s, and rinsed twice in alkaline alcohol for 3 min. Following dehydration in graded ethanol concentration, 95% (15 min) and 100% (twice, 15 min each), samples were cleared with xylene for 1 min and mounted on microscope slides in Canada balsam. Parasites were photographed using a Leica DM1000 microscope (Leica Microsystems, Wetzlar, Germany) at 100 \\u00d7 magnification, and measurements were obtained with the aid of the ImageJ software (LOCI, University of Wisconsin, USA). Our measurement data were presented as the range (mean \\u00b1 SD). For taxonomic identification, we compared worm morphological features and measurement data to previous studies (3, 30\\u201334).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B35\", \"B36\"], \"section\": \"Molecular identification of parasites\", \"text\": \"Genomic DNA was extracted from 10 flukes stored at \\u221220\\u00b0C using a QIAGEN DNeasy\\u2122 tissue kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The gDNA concentrations were evaluated by a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to optimize the amount of gDNA used in PCR reactions. The isolated gDNA was stored at \\u221220\\u00b0C until further use. To identify adult flukes, we selected the nuclear ribosomal internal transcribed spacer region, including part of internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA complete sequence, and part of ITS2. We used the universal primers BD1 (5\\u2032-GTCGTAACAAGGTTTCGGTA-3\\u2032) and BD2 (5\\u2032-TATGCTTAAATTCAGCGGGT-3\\u2032) (35, 36) synthesized by FASMAC Co. Ltd. (Atsugi, Japan). The PCR reactions (25 \\u03bcL) comprised 12.5 \\u03bcL EmeraldAmp MAX Master Mix (Takara Bio, Kusatsu, Japan), 1 \\u03bcL of each primer (10 pmol/\\u03bcL), 20 ng of template DNA, and ddH2O up to 25 \\u03bcL. The PCR was run in a SensoQuest Labcycler 48 (SensoQuest GmbH company, G\\u00f6ttingen, Germany). PCR conditions were pre-denaturation at 94\\u00b0C for 3 min; 30 cycles of denaturation at 94\\u00b0C for 1 min, annealing at 56\\u00b0C for 1 min, and extension at 72\\u00b0C for 2 min; with a final extension at 72\\u00b0C for 5 min. The amplified PCR products were examined by 1% ethidium bromide-stained agarose gel electrophoresis. After cleaning using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), PCR products were sent to Sangon Biotech Co., Ltd (Shanghai, China) for sequencing in both directions.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B37\", \"B38\"], \"section\": \"Bioinformatic analysis\", \"text\": \"We determined homologous sequences using the Basic Local Alignment Search Tool (BLAST) on NCBI. Boundaries between ITS1, 5.8S rRNA, and ITS2 sequences were determined by aligning and comparing our sequence to that of Cyathocotyle prussica (MH521249). We selected 27 Diplostomata sequences for phylogenetic analysis. Selected fragments of the ITS1-5.8S-ITS2 region were assembled by MEGA X (37) and aligned with the Clustal W software (38). Homology percent and pairwise distances were analyzed using the Megalign module of the DNASTAR Lasergene package (v. 7.1.0). The alignments dataset was analyzed using MEGA X to predict the best-fitting nucleotide substitution model based on the Akaike Information Criterion (AIC). Maximum likelihood (ML) and Neighbor-joining (NJ) analyses were performed under the K2 + G model. Bootstrap values of 1,000 resampled datasets were used to estimate the nodal support of the phylogenetic tree. The tree explorer of MEGA X was utilized to visualize the phylogenetic trees. Clonorchis sinensis (MF319655) was used as an outgroup.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B39\"], \"section\": \"Histopathological examination\", \"text\": \"Small intestine tissue samples from each group were fixed in 10% formalin overnight, washed with ddH2O, dehydrated in ascending ethanol concentrations (30\\u2013100%), cleared with xylene, and embedded in paraffin wax. Sections of 3 \\u03bcm thickness were sliced and dewaxed with xylene before rinsing in descending ethanol grades (100\\u201330%) and ddH2O. After staining with hematoxylin and eosin (HE), sections were dehydrated. Slides were scanned, and images were processed using the ImageJ software (LOCI, University of Wisconsin, United States). Intestinal tissue sections were examined for pathological findings and scored for the following: in?ammation with villous atrophy (none = 0, slight = 1, moderate = 2, and severe = 3), in?amed area (mucosa = 1, mucosa and submucosa = 2, and transmural = 3), surface ulceration (none = 0, focal = 1, diffuse= 2, complete loss of surface epithelium = 3, entire surface epithelium and crypt epithelium are lost = 4), and involvement percentage (1\\u201325% = 1, 26\\u201350% = 2, 51\\u201375% = 3, and 76\\u2013100% = 4) (39).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"T1\", \"B40\"], \"section\": \"Quantification of cytokine gene expression\", \"text\": \"Gene expression of eleven cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IFN-\\u03b3, and TGF-\\u03b23) in pigeon intestines and spleen at different time points were evaluated by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). We designed IL-2, IL-4, and IL-5 primers based on conserved regions in genes isolated from related birds available on NCBI. Primers for amplifying other cytokines and internal reference (\\u03b2-actin) genes were synthesized based on published sequences (Table 1). The Applied Biosystems\\u2122 7,500 Real-Time PCR Systems (Applied Biosystem, Bedford, MA, USA) was used to evaluate cytokine expression levels in pigeons during P. vivax infection. The qPCR system was consisted of 0.5 \\u03bcL cDNA (750 ng), 0.5 \\u03bcL of each forward and reverse primers (10 pmol/L), 5 \\u03bcL WizPure\\u2122 qPCR Master (SYBR) (Wizbiosolution, Gyeonggi-do, Republic of Korea), 0.2 \\u03bcL ROX Dye (50X), and ddH2O up to 10 \\u03bcL. The qPCR reaction conditions were as follows: pre-denaturation at 95\\u00b0C for 10 min and 40 cycles of denaturation at 95\\u00b0C for 15 s and annealing at 58\\u00b0C for 1 min. The product specificity was detected by a melting curve program at 95\\u00b0C for 15 s, 60\\u00b0C for 1 min, 95\\u00b0C for 30 s, and 60\\u00b0C for 15 s. Each experiment was conducted in triplicates. The relative expression levels of different cytokines were determined with \\u03b2-actin as the reference gene using the 2\\u2212\\u0394 method (40).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F1\", \"T2\"], \"section\": \"Parasitological examination and worm recovery\", \"text\": \"In preliminary experiments, high inoculation doses (1,000 and 2,000 metacercariae/pigeon) resulted in severe offensive greenish mucoid diarrhea 4 dpi. Pigeon mortality began at 5 dpi, and by 7 dpi, all birds died. However, 500 metacercariae per host infection did not affect pigeon survival, and pigeons only showed mild diarrhea and gradual weight loss. A 100% of challenged birds were infected. The eggs firstly appeared in feces on the fourth day post-infection. Eggs were large, oval-shaped, yellow, and measured 80\\u201390 \\u03bcm long by 50\\u201362 \\u03bcm wide (Figure 1A). On post-mortem examination, we mainly detected flukes in the upper intestine (duodenum and jejunum) of pigeons. Oral infection with 500 metacercariae resulted in the highest worm recovery rate (66.6 \\u00b1 5.2%) at 2 dpi, with an average of 333.3 \\u00b1 19.1 worms/ pigeon. The recovery rate gradually decreased as the infection progressed, with the lowest recovery rate (14.3 \\u00b1 1.7%, 71.7 \\u00b1 6.2 worm/ pigeon) observed in the 28-dpi group. Generally, there was a highly significant difference (p < 0.0001) in the worm recovery at different time points of infection. Multiple comparisons of the number of worms recovered per bird and recovery rate % at each time point showed a significant change between all groups, except for G2 vs. G3 and G4 vs. G5 comparisons (Table 2).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F1\", \"T3\"], \"section\": \"Morphological identification of parasites\", \"text\": \"Based on morphological features, we identified the recovered flukes as Prohemistomum vivax (Sonsino, 1892). The body was undivided into two distinct regions, pyriform or oval, attenuated at both ends and wide at the middle part, concave ventrally. The oral sucker was round and subterminal, leading to a well-developed muscular pharynx. The ventral sucker was well-developed and spherical. The esophagus was short and bifurcated away from the ventral sucker into two intestinal caeca, terminating posterior to the posterior testes. Testes were tandem in position, the anterior one was smooth and ovoid, and the posterior one was quadrilateral. The cirrus pouch was well-developed and on the left side in the caudal region. The ovary was nearly pyramidal, situated laterally between two testes. Vitelline follicles were moderately large, confined in a horseshoe manner around the gonads, and postero-lateral to the tribocytic organ. The caudal appendage and vaginal sphincter were absent. The genital pore was subterminal (Figure 1B). Each organ of P. vivax adults was measured and compared to previous reports (Table 3). Statistical analyses of the measurement data showed significant differences in some parameters between this study and previous studies.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F2\", \"F3\"], \"section\": \"Molecular identification and phylogenetic analysis\", \"text\": \"We determined the pairwise distance of the ITS1-5.8S-ITS2 region of P. vivax with those of 27 Diplostomata trematode sequences in GenBank (Figure 2). The Maximum Likelihood and Neighbor-joining phylogenetic analysis (Figure 3) had the same topology. Based on phylogenetic analysis and the pairwise distance comparison of our sample to the related sequences, our sample (P. vivax) belonged to a clade supported by 100% bootstrap, representing the trematode family Cyathocotylidae. Prohemistomum vivax was most closely related to cyathocotylid metacercariae isolated from common carp (Cyprinus carpio) fish from Hungary (MT668950) with an identity percent of 95.4%. Both samples clustered in one clade supported by a high bootstrap value (96/100%, ML/NJ). This clade clustered with that containing Mesostephanus sp. metacercariae isolates (HM064922-HM064924) from the pumpkinseed (Lepomis gibbosus) fish in Canada, with an identity percent (84.2\\u201389%) to our sample.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F4\", \"T4\", \"F4\", \"F4\", \"F4\", \"F4\", \"T4\", \"T4\"], \"section\": \"Histopathological evaluation\", \"text\": \"Histopathological evaluation of intestinal sections from infected pigeons at different time points showed that P. vivax induced histopathological lesions as early as 2 dpi. The intensity of lesions progressively increased with the infection course (Figure 4, Table 4). Intestinal tissues from groups 1 to 6 revealed a variable degree of villi shortening and thickening or atrophy, and inflammatory cells infiltration in mucosal lamina propria. These alterations gradually accelerated as the infection progressed from 2 to 28 dpi, with the appearance of fused villi at 21 and 28 dpi (Figures 4A\\u2013F). Focal epithelial ulcerations were also detected in intestinal sections at time points following fluke maturity, in 7 and 14 dpi groups (Figures 4C,D). Flukes were seen between the intestinal villi and in the villi interspace without invading the crypt region. Some flukes were attached to the intestinal mucosa, pinching intestinal villi (Figure 4G). Intestinal tissues from uninfected pigeons (NC group) revealed normal long and slender uniform intestinal villi. Lamina propria occasionally showed few inflammatory cells with rounded uniform glands (Figure 4H). We used four parameters to evaluate the histopathological changes in pigeon intestines during infection (Table 4). The values of intestinal histological score gradually increased with the infection course, with the highest score (6) detected in 14-, 21-, and 28-dpi groups. The intensity of inflammatory infiltration and villous alterations gradually accelerated as the infection progressed from 2 to 28 dpi, with the induction of villous fusion at 21 and 28 dpi. Moreover, the involvement percentage increased from up to 25% in the 2-dpi group to 51\\u201375% in the 21- and 28-dpi groups (Table 4).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F5\", \"T5\"], \"section\": \"Cytokine gene expression in intestines\", \"text\": \"To understand how the immune response altered throughout the infection, we compared the gene expression of eleven cytokines in the intestinal tissues of infected and uninfected pigeons using RT-qPCR. Results showed that P. vivax infection significantly increased Th1, Th2, Treg, and inflammatory cytokines gene expression (Figure 5, Table 5). Cytokines transcriptional trends revealed an immunosuppressive response during early infection stages, with decreased expression of some cytokines, such as IL-12, IL-4, IL-5, IL-1, IL-18, and IL-15, at 2 dpi, 4 dpi, or both compared to control. We did not detect any significant change in Th1 cytokines (IFN-\\u03b3, IL-12, and IL-2) gene expression during early infection. Increased IFN-\\u03b3 mRNA signals were detected in small intestine 7 dpi (p = 0.0127) and peaked at 21 dpi (p < 0.0001). IL-12 mRNA signals increased in late infection during days 21\\u201328 dpi. IL-2 significant expression levels were detected at 7\\u201328 dpi, peaking at 7 dpi. IL-10 mRNA levels in the intestine showed a significantly higher expression than the control in late infection from 7 to 28 dpi, peaking at 28 dpi. TGF-\\u03b23 mRNA in intestine was rapidly elevated at 2 dpi (p = 0.0265), peaked at 4 dpi (p = 0.0135), remained significantly high until 7 dpi (p = 0.0418) followed by a non-significant change between 14 and 28 dpi compared to non-infected group. Fluke infection also upregulated Th2 cytokines (IL-4 and IL-5). IL-4 expression was significantly higher in infected groups than in the control group at 14\\u201328 dpi, with the peak value recorded at 21 dpi (7.9-fold-change, p = 0.0044). IL-5 was significantly overexpressed at 7\\u201328 dpi. IL-5 upregulation peaked at 7 dpi and then decreased toward late infection at 28 dpi, from 30 to 2.7 times the control. IL-1 inflammatory cytokine was significantly upregulated at 7 dpi (33-fold-change, p < 0.0001) in infected pigeons compared to control, remained high at 14 dpi (3.6-fold-change, p = 0.0101), and then decreased to basal level. A significantly higher expression level of IL-18 was only detected at 7 dpi (35-fold-change, p = 0.0064). IL-6 transcription level significantly increased in the period from 4 to 28 dpi, with the highest level recorded at 7 dpi (12.8-fold-change, p = 0.0015). IL-15 upregulation was observed at 7\\u201328 dpi, with a gradual decrease toward the end of the experiment.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"F6\", \"T6\"], \"section\": \"Cytokine gene expression in spleens\", \"text\": \"Figure 6 and Table 6 show changes in cytokine mRNA expressions in the spleen of P. vivax-infected pigeons. Increased IFN-\\u03b3 mRNA signals were detected at 7\\u201328 dpi, peaking at 21 dpi (29-fold-change, p = 0.0010). IL-12 mRNA signals significantly increased during days 14\\u201328 PI, with the highest value recorded at 14 dpi and the lowest detected at 28 dpi. IL-2 cytokine expression significantly increased at 7\\u201328 dpi. A significant increase in IL-10 mRNA levels was detected late at 14 dpi, with the peak (10.8 times that of the control group) occurring at 21 dpi (p = 0.0036). TGF-\\u03b23 mRNA was significantly elevated throughout the infection from 2 to 28 dpi. However, the fold change of upregulation peaked at 2 dpi (31.9-fold-change, p < 0.0001) and gradually decreased as the infection progressed. Th2 cytokines expression significantly increased at 7\\u201328 dpi for IL-5 and 14\\u201328 dpi for IL-4. The peak expression level was detected at 21 dpi for IL-4 and IL-5. IL-1 cytokine was significantly upregulated at 14\\u201328. Significantly higher expression levels of IL-6 and IL-18 were found at 7\\u201328 dpi. IL-15 upregulation was observed at 4\\u201328 dpi.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"T7\"], \"section\": \"Correlation analysis of Th1/Th2/Treg cytokines\", \"text\": \"Pearson's correlation analysis was conducted on each pair of cytokines based on relative expression quantity (RQ) from infected groups (Table 7). In the intestine, this analysis showed a significant positive association between IFN-\\u03b3 mRNA expression and IL-2 (r = 0.86, p < 0.0001) and Th2 cytokine (IL-4: r = 0.61, p = 0.0072) as well as between IL-2 and IL-5 (r = 0.64, p = 0.0042). IL-10 gene expression was significantly associated with both Th1 (IFN-\\u03b3: r = 0.48, p = 0.0461; IL-12: r = 0.92, p < 0.0001) and Th2 cytokines (IL-4: r = 0.49, p = 0.0380). TGF-\\u03b23 relative mRNA expression demonstrated a significant negative correlation to the other cytokines (IFN-\\u03b3: r = \\u22120.65, p = 0.0032; IL-12: r = \\u22120.76, p = 0.0002; IL-4: r = \\u22120.70, p = 0.0011; IL-10: r = \\u22120.79, p < 0.0001).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"T7\"], \"section\": \"Correlation analysis of Th1/Th2/Treg cytokines\", \"text\": \"In spleens, a strong positive correlation was also detected between IFN-\\u03b3 and Th2 cytokine expressions (IL-4: r = 0.86, p < 0.0001; IL-5: r = 0.82, p < 0.0001). IL-12 mRNA was positively correlated to Th2 cytokines (IL-4: r = 0.66, p = 0.003; IL-5: r = 0.51, p = 0.0315) and other Th1 cytokines (IFN-\\u03b3: r = 0.61, p = 0.0068; IL-2: r = 0.50, p = 0.0352). A strong positive correlation was detected between IL-4 and IL-5 (r = 0.94, p < 0.0001). IL-10 expression in spleen was positively correlated with both Th1 and Th2 cytokines (IFN-\\u03b3: r = 0.76, p = 0.0002; IL-12: r = 0.66, p = 0.0030; IL-4: r = 0.91, p < 0.0001; IL-5: r = 0.83, p < 0.0001). The relative mRNA expression of TGF-\\u03b23 was negatively correlated to other cytokines (IFN-\\u03b3: r = \\u22120.83, p < 0.0001; IL-12: r = \\u22120.62, p = 0.0056; IL-4: r = \\u22120.84, p < 0.0001; IL-5: r = \\u22120.68, p = 0.0021; IL-10: r = \\u22120.86, p < 0.0001), in Table 7.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"T8\"], \"section\": \"Comparing cytokine expression in the intestine and spleen of P. vivax-infected pigeons\", \"text\": \"Table 8 compares relative cytokine expressions between the intestine and spleen of P. vivax-infected pigeons. Our findings revealed a significant difference in the relative quantity of cytokines between the two organs throughout the infection. Th1 (IFN-\\u03b3 and IL-12), IL-4, TGF-\\u03b23, and IL-15 cytokines had significantly higher expression in the spleen than in the intestine. However, at 7, 21, and 28 dpi, IL-2 and IL-10 levels in the intestine were significantly higher than those in the spleen. Other cytokines (IL-5, IL-1, IL-6, and IL-18) were significantly higher in the intestine at early infection but became higher in the spleen as the infection progressed. IL-5 levels were higher in the intestine than in the spleen at 2\\u201314 dpi, with a significant difference at 7\\u201314 dpi. Then, at 21\\u201328 dpi, IL-5 levels in the spleen were higher than in the intestine. The intestine had significantly higher expression levels of IL-1 at 7 dpi, IL-6 at 4\\u20137 dpi, and IL-18 at 2 and 7 dpi, while the spleen had higher levels of all three cytokines at 14\\u201328 dpi.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B2\", \"B5\", \"B7\", \"B17\", \"B34\", \"B17\", \"B17\", \"B18\"], \"section\": \"Discussion\", \"text\": \"In this study, the morbidity and fatality in infected pigeons depended on the infection dose. The ingestion of 1,000 or more metacercariae resulted in the death of all birds within a week of infection, while 500 metacercariae inoculum did not cause fatalities in pigeons during the studied period of 28 days. Similarly, the severity of the clinical signs and the mortality in Neodiplostomum seoulense-experimentally infected mice was proportional to the metacercariae inoculum. Mice receiving 1,000 N. seoulense metacercariae died within 16 days post-infection. The possible causes of death might be excessive fluid loss and malnutrition due to malabsorption, mucosal bleeding, and persistent diarrhea (2). The earlier death in our study compared to that recorded with N. seoulense might be related to the difference in host and parasite species. We showed that metacercariae could successfully develop into adult P. vivax in the intestine of pigeons. This result agreed with previous studies (5, 7). P. vivax characteristic eggs were firstly detected in pigeon feces 4 days post-infection, in agreement with previous results in mice and rats (17, 34). Mahfouz et al. (17) suggested that the parasite's early growth may necessitate a rich supply of nutrients. Thus, it is possible to detect the parasite engulfing the epithelial villi. The upper intestine was the primary habitat of P. vivax in the final host (pigeons), consistent with results reported in P. vivax-infected mice (17, 18).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B3\", \"B30\", \"B32\", \"B34\", \"B32\", \"B33\", \"B42\", \"B3\", \"B32\", \"B33\", \"B34\", \"B42\"], \"section\": \"Discussion\", \"text\": \"The recovered flukes were confirmed as P. vivax based on combined morphological and molecular identification. Although, there was a significant difference between our sample measurements and that of previous studies in some parameters, our sample matched all morphological characters of P. vivax reported in previous studies (3, 30, 32\\u201334). Our sample was smaller than that recorded by Fahmy and Selim (32) and El-Nafffar et al. (33). This difference might be related to the different biological environments in the intestine of variable hosts (animals and birds) (42) and the difference between experimental and natural infection. For example, we compared measurements of P. vivax recovered from human (3), naturally infected dogs (32), experimentally infected dogs and cats (33), and experimentally infected rats (34) Similarly, Fahmy et al. (42) showed that P. vivax recovered from dogs were larger than that collected from buff-backed herons. We successfully amplified the ITS1, 5.8 S rRNA, and ITS2 region from P. vivax for the first time. In the phylogenetic tree, our sample (P. vivax) belonged to the family Cyathocotylidae clade. P. vivax was closely related to cyathocotylid metacercariae recovered from Hungarian common carp fish (95.4% homology) and clustered in one clade with Mesostephanus sp. metacercariae, which might represent the subfamily prohemistominae clade. These findings agreed with our sample morphological description and known taxonomy.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B17\", \"B7\", \"B17\", \"B43\", \"B44\", \"B2\", \"B45\", \"B17\", \"B2\", \"B46\", \"B47\"], \"section\": \"Discussion\", \"text\": \"In histopathological examination, we found P. vivax flukes between the intestinal villi and never invaded the crypt region, similar to findings in mice (17). P. vivax caused histopathological alterations as early as 2 dpi. Furthermore, lesion intensity and intestinal histological score gradually increased with the infection course, with the highest score (6) detected on 14, 21, and 28 dpi. Intestinal tissue lesions included shortening and thickening or atrophy of villi, villous fusion, inflammatory cell infiltration in mucosal lamina propria, and focal epithelial ulcerations. Similar pathological changes were seen in the upper intestine of P. vivax-infected pigeons and mice (7, 17), Heterophyes heterophyes-infected mice (43), Echinostoma malayanum-infected hamsters, rats, mice, and gerbils (44), Metagonimus yokogawai-infected cats, N. seoulense-infected rats and mice, Pygidiopsis summa-infected rats and mice, and Gymnophalloides seoi-infected mice, reviewed by Chai (2). The observed pathological alterations might be attributed to the parasite's direct effect and the host's response. Intestinal flukes, including P. vivax, can induce mechanical and chemical damage in the host gut. P. vivax possesses a spiny tegument and well-developed adhesive tribocytic organ (45), which may directly affect parasite pathogenesis due to irritation of the host mucosa (17). Additionally, the tribocytic organ of diplostomidean flukes can pierce the host villi and secrete alkaline phosphatase that can lyse the villi (2). Early studies proposed that frequent movement and rotation of the trematode anterior part injury the intestinal mucosa and result in mechanical pressure on adjacent villi (46). Chai (47) suggested that the villi pressure atrophy in M. yokogawai-infected rat intestine might be related to the accumulation of mucus and gases in the gut lumen.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B17\", \"B48\", \"B49\", \"B50\", \"B25\", \"B25\", \"B26\"], \"section\": \"Discussion\", \"text\": \"Previous reports have supported the induction of host protection mechanisms against P. vivax, activating the humoral and cellular host immune responses (17). Helal et al. (48) observed a massive influx of eosinophils and mast cells in the intestines of infected rats. Still, no studies have investigated the molecular pathology and the immune effector mechanism of intestinal infection with P. vivax. The immune response is regulated by CD4 T cells that are primarily classified into Th1 or Th2 T cells based on the released cytokines. Th1 cells mainly release IFN-\\u03b3 and IL-12, while Th2 cells release IL-4, IL-5, IL-9, IL-10, and IL-13 (49, 50). These cell subsets induce different and counterregulatory immune responses. Th2 response cytokines regulate the host protection, whereas Th1 responses are related to chronic infections (25). Nevertheless, the preferential stimulation of Th1 and Th2 cells during parasite infections depends on many variables, including host genetic nature, parasite species, infection route and stage, and acute or chronic infection (25, 26).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B50\", \"B17\", \"B51\", \"B52\", \"B53\", \"B50\", \"B54\"], \"section\": \"Discussion\", \"text\": \"This study demonstrated that P. vivax infection significantly impacted Th1, Th2, Treg, and inflammatory cytokines gene expression. The cytokine transcriptional profile indicated an immunosuppressive response in the early stages, a mixed Th1/Th2 response as the infection progressed, and a late tilting toward Th1/Treg response. At early infection (2\\u20134 dpi), the expression of some cytokines was generally lower than in controls, implying an immunosuppressive condition to facilitate parasite colonization. This early immunosuppression state and cytokine decline were also observed during Fasciola gigantica infection and were attributed to the overexpression of the immunosuppressive cytokine TGF-\\u03b2 at 3 dpi (50). In agreement with this hypothesis, our study showed that TGF-\\u03b23 mRNA was rapidly elevated in the intestine at 2 dpi and remained significantly high until 7 dpi, followed by a non-significant change at 14\\u201328 dpi. Although TGF-\\u03b23 mRNA in the spleen was significantly high throughout the infection period, the fold change of expression peaked at 2 dpi and gradually diminished as the infection progressed. These results suggest that TGF-\\u03b23 might play a role in immunosuppression and parasite colonization during early P. vivax infection. Similarly, semi-quantitative RT-PCR revealed an upregulation in TGF-\\u03b2 expression in P. vivax-infected mice intestines during the examined first 6 days of infection (17). TGF-\\u03b2 was also overexpressed during various parasitic infections, such as Schistosoma japonicum (51), Echinococcus granulosus (52), and Heligmosomoides polygyrus (53). We detected a significant negative correlation between TGF-\\u03b23 and other cytokines in both intestines and spleen of infected groups. Although there was no significant difference, TGF-\\u03b2 was also inversely related to other cytokines during F. gigantica infection (50). This negative correlation might be related to the immunosuppressive role of TGF-\\u03b2. TGF-\\u03b2 can suppress T-cell proliferation, cytokine production and cytotoxicity, IFN-\\u03b3 production, MHC class II expression, and reactive oxygen intermediates and nitric oxide release from activated macrophages (54).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B26\", \"B26\", \"B55\", \"B56\", \"B26\", \"B57\", \"B58\", \"B59\", \"B60\", \"B50\"], \"section\": \"Discussion\", \"text\": \"Here, as P. vivax infection progressed 7\\u201328 dpi, a mixed Th1 and Th2 responses developed in agreement with results of Shin et al. (26), who showed that mixed Th1 and Th2 responses shared in the activation of humoral and cellular immune reactions throughout N. seoulense infection in mice. P. vivax infection upregulated Th1 (IFN-\\u03b3, IL-12, and IL-2) and Th2 cytokines (IL-4 and IL-5). IFN-\\u03b3 is released from Th1 cells and is linked to proinflammatory cytokines production, inhibiting Th2 responses (26). IL-12 is critical for enhancing cell-mediated immune responses. IL-12 and IL-12R-deficient humans and mice had impaired immune responses and were vulnerable to intracellular pathogen infections. IL-12 plays an important role in Th cell differentiation into the Th1 subset. IL-12 also stimulates IFN-\\u03b3 release, in synergy with IL-2 or IL-18, and T and NK cell proliferation and cytolytic activity (55). IL-2 was the first cytokine to be molecularly cloned and was identified as a T cell growth factor vital for T cell proliferation and effector and memory cell generation. Subsequent research showed that IL-2 is necessary for maintaining Treg cells, and its absence results in an intense Treg cell deficiency and autoimmunity because IL-2 encourages Treg cell generation, survival, and activity. Therefore, IL-2 serves two contradictory roles: enhancing conventional T cells to boost immune responses and maintaining Treg cells to control immune responses (56). We found that IFN-\\u03b3 was upregulated in both the intestine and the spleen (at 7 dpi) before IL-12, which may be contradictory. Overexpression of IFN-\\u03b3 also proceeded IL-12 upregulation in the spleen and mesenteric lymph node of N. seoulense-infected mice, but not in the small intestine (26). Although IL-12 is a major inducer of IFN-\\u03b3, IL-2 also significantly impacts IFN- expression. These cytokines (IL-12 and IL-2) can induce IFN-\\u03b3 expression independently. However, they act synergistically to induce large amounts of IFN-\\u03b3, particularly from NK cells (57). At 7 dpi, we found IL-2 upregulation in the intestine and spleen, with 29- and 17-fold increases compared to controls, respectively. The overexpressed IL-2 might have induced IFN-\\u03b3 production either independently or in synergy with the low amount of IL-12 expressed during early infection. IFN-\\u03b3 and IL-12 expression is coordinated, meaning that IL-12 induces IFN-\\u03b3, and IFN-\\u03b3 induces IL-12 (58). Therefore, when the expression of IFN-\\u03b3 increased, IL-12 was upregulated, which in turn exaggerated IFN-\\u03b3 release. IL-4 is produced by Th2 cells and is essential for Th2 cell differentiation, as well as B cell activation, enabling IgE production. This cytokine plays a crucial role in parasite infections, promoting the host's resistance to parasite invasion (59). IL-5 is a cytokine that is involved in the Th2-type inflammatory response in the host and triggers eosinophil production and stimulation (60). In this study, Th1 and Th2 cytokine expressions were positively correlated, consistent with the findings of Shi et al. (50). This positive correlation might be related to the antagonistic functions of both responses and could be essential for immune homeostasis.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B20\", \"B24\", \"B26\", \"B25\", \"B25\", \"B25\", \"B26\", \"B26\", \"B46\", \"B26\"], \"section\": \"Discussion\", \"text\": \"Intestinal nematodes, such as Heligmosomoides polygyrus, Trichuris muris, and Nippostrongylus brasiliensis, can induce strong Th2 responses (20, 24). On the contrary, intestinal trematodes, such as N. seoulense, can activate mixed Th1 and Th2 responses, but Th1 cytokine production (IFN-\\u03b3 and IL-12) was the more potent among them (26). This result agreed with observations for Echinostoma caproni (25), which mainly triggered Th1 responses (elevated IFN-\\u03b3 and IL-12) while also increasing Th2 cytokines (IL-5 and to a lower extent, IL-4). A single injection of anti-IFN-\\u03b3 antibodies to E. caproni-infected mice significantly reduced worm burden, suggesting the IFN-\\u03b3 role in establishing chronic echinostomiasis (25). Host fatalities were recorded in N. seoulense and P. vivax but not E. caproni infections. Thus, chronic infection is linked to a Th1 response (25), while mixed Th1 and Th2 responses may contribute to immunopathogenesis that results in host injury and worm expulsion (26). In N. seoulense-infected mice spleens and intestines, numbers of macrophages, activated either by Th1 or Th2 cells, were increased at 14\\u201328 dpi (26), the expected period for worm expulsion (46). In vitro, rat macrophages eliminated N. seoulense through antibody-dependent cellular cytotoxicity. As a result, Shin et al. (26) concluded that combined Th1 and Th2 responses could result in extensive macrophage infiltration in the infected rat spleen and small intestine, enhancing worm ejection and host damage. Although this conclusion might be functional during the infection with P. vivax, a diplostomidean fluke related to N. seoulense, more studies are required to assess the role of macrophages in P. vivax.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B26\", \"B27\", \"B50\", \"B61\", \"B62\", \"B63\", \"B64\", \"B65\", \"B65\", \"B66\", \"B50\"], \"section\": \"Discussion\", \"text\": \"Treg cytokine IL-10 mRNA levels were significantly upregulated at 7\\u201328 dpi in the intestine and 14\\u201328 dpi in the spleen. This result agreed with IL-10 upregulation in other helminthic infections, such as N. seoulense (26), E. caproni (27), Fasciola gigantica (50), Toxocara canis (61), Ostertagia ostertagi (62), Strongyloides venezuelensis (63), and Trichinella spiralis (64). IL-10 is an anti-inflammatory cytokine released from Treg cells that can reduce the immunopathological damage in the host by suppressing parasite-induced inflammation (65). IL-10 is also critical for Th2 response and induces T cell response shifting to the Th2 type while acting as a suppressive regulator of IL-12 (Th1) and inhibiting Th1 cell differentiation (65, 66). We suggest that IL-10 expression increased during P. vivax infection to counteract the high levels of inflammatory cytokines and maintain the Th1/Th2 balance and immune homeostasis, thereby protecting the host from excessive inflammation and tissue pathology. In our study, there was a positive correlation between IL-10 and Th1 and Th2 cytokines, consistent with the results in F. gigantica-buffaloes recorded by Shi et al. (50). Increasing IL-10 levels with the concurrent upregulation of Th1 and Th2 cytokines suggest that IL-10 is required for the Th2 response to P. vivax infection. Moreover, IL-10 overexpression follows Th1 response rise to hinder the overexaggerated Th1 cell activity, maintaining Th1/Th2 balance and immune homeostasis.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B50\", \"B67\", \"B67\", \"B68\", \"B68\", \"B69\", \"B70\", \"B69\", \"B71\"], \"section\": \"Discussion\", \"text\": \"IL-1 inflammatory cytokine was significantly upregulated at 7\\u201314 dpi in the intestine and 14\\u201328 dpi in the spleen. Similarly, the cytokine IL-1\\u03b2 mRNA was overexpressed in the livers of F. gigantica-infected buffaloes (50). We also detected a significantly higher IL-18 expression level at 7 dpi in the intestine and 7\\u201328 dpi in the spleen, in agreement with Alhallaf et al. (67), who showed elevated IL-18 secretion in humans and mice infected with Trichuris nematodes. The IL-1 family of cytokines (IL-1\\u03b1, IL-1\\u03b2, and IL-18) is released from innate immune cells, such as macrophages, monocytes, dendritic cells, endothelial, and epithelial cells, and are essential for initiating and enhancing innate and adaptive immune responses, resisting microbial infections, regulating immune response (67, 68). IL-18 can promote Th2-type immune responses via stimulating IL-4 and IL-13 production from basophils in the presence of a second stimulus (IL-3). Additionally, IL-18 plays a vital role in priming NK cells for IFN-\\u03b3 production and increases its secretion in response to IL-12 (68). IL-1\\u03b1 and IL-1\\u03b2 have many immunomodulatory roles, including activating monocytes and macrophages, stimulating B cell proliferation and differentiation in collaboration with IL-4 and IL-6, and potentiating antibody production (69). Some studies suggest that IL-1\\u03b1 and IL-1\\u03b2 have a physiological impact on Th cell response development. IL-1 can enhance T cell proliferation by stimulating the transcription of IL-2 receptor (70). Moreover, lymph node cells from IL-1\\u03b1 and \\u03b2-deficient mice failed to differentiate into Th2 cells in the presence of IL-4. Thus, IL-1\\u03b1 and IL-1\\u03b2 are required, along with IL-4, for Th2 response development. In vivo, IL-1\\u03b1 and IL-1\\u03b2 were involved in establishing Th2-mediated resistance (expulsion) to nematodes, where IL-1\\u03b1 and IL-1\\u03b2-deficient mice were vulnerable to chronic Trichuris muris infection, with impaired Th2 responses (69). IL-1 has also been essential for IL-12-induced Th1 cell growth (71). Concurrent overexpression of IL-1 and IL-18 with Th1 and Th2 cytokines in P. vivax infection might support previous suggestions for their role in developing Th1 and Th2 responses.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B27\", \"B72\", \"B27\", \"B73\", \"B27\"], \"section\": \"Discussion\", \"text\": \"We demonstrated that IL-6 transcription levels significantly increased in the intestine at 4-28 dpi and in the spleen at 7-28 dpi. Similar to our results, E. caproni-infected rats and mice showed a significant increase in IL-6 in the spleen and intestine (27). IL-6 activates T helper cells, regulates T cell resistance to apoptosis, and modulates the Treg/Th17 cells balance (72). IL-6 acts as a myokine, i.e., a cytokine released from muscle during contraction (27, 73). IL-6 expression was more marked in the intestine of E. caproni-infected rats (resistant host) compared to mice (compatible host), suggesting the possible role of IL-6 in intestinal motility in response to intestinal trematode (27). IL-6 upregulation might alter intestinal smooth muscle motility, hindering worm attachment and feeding in the intestine, thus helping worm expulsion during P. vivax infection. Additionally, the high expression of Th1 cytokines (IL-1, IL-18, and IL-6), indicating acute inflammatory response, might be linked to the severe inflammatory cell infiltrations observed in the pigeon intestine.\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B74\", \"B75\", \"B76\", \"B75\", \"B75\", \"B77\", \"B77\", \"B81\", \"B82\", \"B83\"], \"section\": \"Discussion\", \"text\": \"We observed IL-15 upregulation at 7\\u201328 dpi in P. vivax-infected pigeons' intestines and 4\\u201328 dpi in spleens. IL-15 is a proinflammatory cytokine involved in the development, proliferation, and activation of several lymphocyte lineages (74). This cytokine acts as a pleiotropic lymphokine that is not expressed in T cells but is derived from many other cells and tissues, including skeletal tissue, muscle, placenta, lung, kidney, heart, epithelial cells, fibroblasts, and monocytes (75). IL-15 belongs to the common cytokine receptor common gamma chain (\\u03b3c) family of cytokines (with IL-2, IL-4, IL-7, IL-9, and IL-21), playing essential roles in innate and adaptive immunity. IL-15 receptor (IL-15R) shares the \\u03b2 chain and \\u03b3 chain of IL-2R. Therefore, IL-15 shares some immunological activities with IL-2, such as stimulating CD4+ and CD8+ T-cell proliferation and activation, T helper cells differentiation, B cells antibodies synthesis, natural killer (NK) cell proliferation and maintenance, and CD8+T cells and NK cells cytolytic activity (76). However, IL-15 activities on T cell-mediated immune responses are distinct from those of IL-2 because IL-15 uses a different \\u03b1-chain from IL-2R and expresses a specific receptor (IL-15RX). IL-15 can suppress the IL-2-mediated T cell responses and Fas-mediated activation-induced cell death (AICD) process. In contrast to IL-2, IL-15 has little effect on Treg activity, but it can compromise Treg function by acting on CD4+T and CD8+T cells (75). IL-15 can boost both Th1 responses by enhancing IFN-\\u03b3 and TNF-\\u03b1 production and Th2 responses by enhancing IL-4 and IL-5 release (75, 77). Studies have revealed that IL-15 might play a role in resisting intracellular protozoan infections, including Leishmania braziliensis, Cryptosporidium, Plasmodium, and Toxoplasma gondii, by influencing Th1 or Th2 response development (77\\u201381). Although little is known about the role of IL-15 in helminth infections, some studies indicated that IL-15 plays a role in the immune response against helminth parasites similar to that played in protozoan parasite infections. The mRNA levels of IL-15 in the immunized group slightly exceeded those found in the O. ostertagi-infected group (82). Filarial nematode (Setaria equina) excretory-secretory antigens stimulated IL-15 production in mice (83).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B27\"], \"section\": \"Discussion\", \"text\": \"The immune response was quite different between the intestine and spleen tissues of P. vivax-infected pigeons. The Th1 response (IFN-\\u03b3 and IL-12) in the intestines appeared more persistent until the end of the experiment, while Th2 cytokine (IL-5) expression fold change peaked at 7 dpi and then decreased toward late infection at 28 dpi. However, the general trend of both Th1 and Th2 cytokines in the spleen was nearly similar, significantly overexpressed at 7\\u201328 or 14\\u201328 dpi and peaking at 14 or 21 dpi. Similarly, Trelis et al. (27) recorded a marked Th1 response (IFN-\\u03b3) in the intestine and a mixed Th1/Th2 phenotype in the spleen of E. caproni-infected high compatible host (mice).\"}, {\"pmc\": \"PMC9516004\", \"pmid\": \"\", \"reference_ids\": [\"B84\", \"B84\", \"B84\", \"B26\"], \"section\": \"Discussion\", \"text\": \"On comparing the relative cytokine quantity in the intestine and spleen, we observed a significant difference in the cytokine expression levels between the intestine and the spleen throughout the infection. Intestinal epithelial cells represent the first barrier of enteric immunity. Helminths cause intestinal tissue damage during feeding, stimulating the release of cytokine alarmins, such as IL-25, IL-33, and thymic stromal lymphopoietin. These alarmins triggers innate lymphoid 2 cells, a major source of the Th2 cytokines (IL-5 and IL-13). As a result, IL-5 and IL-13 levels can rise within hours of helminth infection (84). These findings could explain the early higher expression of IL-5 in the intestine than in the spleen. The lamina propria provides an influential immune cell population, quickly activated after intestinal helminth infection (84). Inflammatory cell infiltration in the lamina propria was detected in the intestine of P. vivax-infected pigeons as early as 2 dpi, which could be related to the higher expression of inflammatory cytokines (IL-1, IL-6, and IL-18) in the intestine during early infection. Additionally, helminth excretory/secretory products and activated dendritic cells circulate to several organs, including the spleen, where they contribute to systemic response development. The spleen is a major source of serum immunoglobulins and Th2 cells. Two weeks after infection with H. polygyrus, splenomegaly was observed, with augmented type 2 cytokine expression. Thus, the intestinal mucosal response develops first and is accompanied by higher Th2 cytokine levels, whereas the systemic response (spleen) quickly follows the initial enteric response (84). This hypothesis could explain IL-5, IL-1, IL-6, and IL-18 increased expression in the spleen after 2 weeks of infection following the upregulation in the intestine. Although the spleen expressed more IFN-\\u03b3, IL-12, IL-4, TGF-3\\u03b2, and IL-15 than the intestine, the difference was mainly significant after 7 dpi, possibly after the systemic response initiation. Even though not statistically analyzed, the spleen of N. seoulense-infected mice had a higher relative quantity of IFN-\\u03b3 and IL-4 than the intestine (26), similar to our results. The type 2 immune response at the site of intestinal helminth infection maintains the stimulates of Th2 effector cells and T regulatory cells, which may lead to higher IL-10 levels in the intestine than in the spleen. This upregulation was maintained in the intestine till 28 dpi, perhaps to hinder the increased inflammatory cytokine expressions in the intestine and protect the host from pathological damage. Like IL-10, IL-2 was highly expressed in the intestine compared to the spleen, which might be related to IL-2 role in maintaining Treg cell survival and activity.\"}]"
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