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Hericium erinaceus polysaccharide facilitates restoration of injured intestinal mucosal immunity in Muscovy duck reovirus-infected Muscovy ducklings
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Hericium erinaceus polysaccharide facilitates restoration of injured intestinal mucosal immunity in Muscovy duck reovirus-infected Muscovy ducklings Yijian Wu a,b , Huihui Jiang a,c , Erpeng Zhu a,b , Jian Li a,b , Quanxi Wang a,b , Wuduo Zhou a,c , Tao Qin a,b , Xiaoping Wu a,c , Baocheng Wu a,b , Yifan Huang a,b,∗ a
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou 350002, People’s Republic of China c University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fuzhou 350002, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 14 June 2017 Received in revised form 13 September 2017 Accepted 22 September 2017 Available online xxx Keywords: Muscovy duck reovirus Hericium erinaceus polysaccharide Duodenum Mucosal immunity Restoration
a b s t r a c t To elucidate the effect of Hericium erinaceus polysaccharide (HEP) on the intestinal mucosal immunity in normal and Muscovy duck reovirus (MDRV)-infected Muscovy ducklings, 1-day-old healthy Muscovy ducklings were pretreated with 0.2 g/L HEP and/or following by MDRV infection in this study, duodenal samples were respectively collected at 1, 3, 6, 10, 15 and 21 day post-infection, tissue sections were prepared for observation of morphological structure and determination of intestinal parameters (villus height/crypt depth ratio, villus surface area) as well as counts of intraepithelial lymphocytes (IELs), goblet cells, mast cells. Additionally, dynamics of secretory immunoglobin A (sIgA), interferon-␥ (IFN-␥) and interleukin-4 (IL-4) productions in intestinal mucosa were measured with radioimmunoassay. Results showed that HEP significantly improved intestinal morphological structure and related indexes, and significantly inhibited the reduction of intestinal mucosal IELs, goblet cells and mast cells caused by MDRV infection. Furthermore, HEP significantly increased the secretion of sIgA, IFN-␥ and IL-4 to enhance intestinal mucosal immune functions. Our findings indicate that HEP treatment can effectively repair MDRV-caused injures of small intestinal mucosal immune barrier, and improve mucosal immune function in sick Muscovy ducklings, which will provide valuable help for further application of HEP in prevention and treatment of MDRV infection. © 2017 Published by Elsevier B.V.
1. Introduction Muscovy duck reovirus (MDRV) causes an infectious disease in Muscovy ducklings with a high morbidity and mortality. Acute cases of MDRV infections were first described in South Africa in 1950 [1], and the virus was isolated from sick ducks in France in 1972 [2], and later in Israel, Germany and other regions [3,4]. In China, MDRV infection first appeared in 1997, and usually named as “liver white spot disease”, which is characterized clinically by soft foot, diarrhea, stunted growth and white necrotic foci in liver and spleen [2,5,6], resulting in huge economic losses in Muscovy
∗ Corresponding author at: Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou 350002, People’s Republic of China. E-mail address: [email protected] (Y. Huang).
duck industry. The etiological agent of “liver white spot disease” was isolated and first defined as MDRV by our group in 2001 [5], which belongs to the family Reoviridae, genus Orthoreovirus. Further studies have showed that MDRV infection induces serious immunosuppression in sick Muscovy ducklings, including severe damages to central immune organs (bursa of Fabricius, thymus), peripheral immune organs (spleen) as well as the associated cellular and humoral immunoresponses [7–10]. Reduction of the immune protections from vaccines and other pathogens secondary to MDRV infection often aggravate this disease due to the suppression of immune function in sick Muscovy ducklings. Intestinal mucosal immunity in animals especially in poultry plays an important part in the body’s immune system. We have previously found that MDRV infection causes serious damage to intestinal mucosa and intestinal mucosal immune system, as well as absorption disorders in Muscovy ducklings [11,12], thus exacerbating the development and outcome of this disease. Further studies have showed that
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MDRV-caused mucosal immunologic injuries are probably associated with intestinal mucosal inflammation, bleeding, degeneration, as well as necrosis, apoptosis and large decreases of lymphocytes in mucosa-associated immune tissues (cecal tonsils) [7,8]. As a kind of immunosuppressive disease with high mortality rate, MDRV infection has attracted great attentions of researchers. Since the genome of MDRV consists of ten segments of doublestranded RNA (dsRNA) and is prone to genetic variation and recombination, the diversifications of disease patterns as well as expansion of MDRV infection spectrum and epidemic areas generally occur, thus greatly increasing the difficulties of prevention and control for MDRV. Domestic researchers have developed MDRV attenuated vaccine, inactivated vaccine and genetic engineering vaccine to prevent the disease [13–15], however, whether the vaccines are able to provide enough safe protection remains to be further verified. Currently, there are no commercially available agents for treatment of MDRV-induced immunosuppression. During the course of developing efficient approaches to deal with immunosuppression caused by MDRV, our group has performed long-term researches. Many traditional Chinese medicine polysaccharide extracts have immunomodulatory and immune-enhancing functions with no drug resistance and residue problems, from which to find potential candidates to improve or cure MDRV-caused immunosuppressive injuries will be of great significance. Hericium erinaceus polysaccharide (HEP) is a kind of polysaccharide extracts from H. erinacues fruiting body, mycelia and mycelial fermentation broth, which is naturally non-toxic and is one of the most important active substances in H. erinacues [16]. HEP has many biological activities, such as enhancing immune functions, scavenging free radicals, as well as facilitating regeneration and repair of gastrointestinal mucosa [17–20]. Considering these advantages, we have previously exploited HEP to treat MDRV infection and have found that HEP pre-treatment could efficiently protect Muscovy ducklings from MDRV infection, including alleviating clinical symptoms, reducing morbidity and mortality and promoting recovery of sick Muscovy ducklings [12,21]. However, the main mechanism remains unclear and needs to be further studied. To elucidate the effect of HEP on the intestinal mucosal immunity in MDRV-infected Muscovy ducklings, in the present study, healthy Muscovy ducklings were pretreated with HEP and then infected with MDRV based on the established MDRV natural infection model, and then morphological structure and related parameters of small intestinal mucosa, counts of mucosal immunocytes such as intraepithelial lymphocytes (IELs), goblet cells and mast cells, as well as sIgA, Th1 cytokine (IFN-␥) and Th2 cytokine (IL-4) productions in intestinal tissues were determined. This study will provide valuable help for elucidating the immunomodulatory mechanism of HEP on intestinal mucosal immunosuppression, and for further application of HEP in prevention and treatment of MDRV infection.
2. Materials and methods 2.1. Materials and chemicals H. erinacues were purchased from Huishihao Pharmacy in Fujian Province of China. Muscovy duck reovirus (MDRV) YB strain was isolated and stored by our laboratory [5]. Slides, Toluidine blue staining kit and Glycogen periodic acid-Schiff (PAS) staining kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The radioactive immunoassay kit for secretory immunoglobulin A (sIgA), interferon-␥ (IFN-␥) and interleukin4 (IL-4) were purchased from Beijing Huaying Biotechnology Research Institute (Beijing, China). All the other chemicals used in this study were of analytical grade.
2.2. Preparation and characterization of H. erinaceus polysaccharides The H. erinaceus polysaccharides (HEP) were extracted from fruiting bodies of H. erinaceus according to the procedures described in our previous study [22]. Briefly, 1000 g of dried H. erinaceus was crushed into small pieces, soaked with 95% ethanol, and refluxed twice in water bath of 80 ◦ C. After drying, the drug was decocted with 20-fold volume water 3 times each for 30 min. The mixture was filtrated through two-layer gauze, and the retentate was concentrated into 1000 mL and then mixed with 95% (v/v) ethanol to yield a 90% ethanolic solution. Finally, the precipitation was lyophilized to obtain the crude HEP with a vacuum freezedrying machine (Model LGJ-25, Dongxing Machinery Industry Co., Ltd. Shamen City). Proteins in the crude HEP were eliminated with Sevage method [23], and the products were further purified by DEAE-52 cellulose and Sephadex G-100 (1.5 cm × 200 cm). The obtained main polysaccharides fraction was subjected to determination of HEP content by the phenol-sulfuric acid method [24,25], and then freeze-dried to get the purified HEP, which was then used in our following biological studies. Besides, as previously described, the purified HEP was subject to Fourier-transform infrared spectroscopy (FTIR) analysis, and the average molecular weight (MW ) of the HEP was measured through gel permeation chromatography (GPC) method, and the monosaccharide composition of the HEP was determined by high-performance lipid chromatography (HPLC) [17,19,22].
2.3. Animals and experimental treatments Two hundred 1-day-old healthy Muscovy ducklings weighing 44.85 ± 9.23 g were provided by the Putian (Guangdong) Wen’s poultry Co., Ltd (Fujian, China), and randomly divided into four groups, blank control group (BCG), HEP control group (HCG), cohabitation infection control group (CICG) and HEP prevention group (HPG), each consisting of 50 ducklings. BCG was only treated with physiological saline solution (0.9%, w/v), once daily during the whole testing period (till to 21-day post-infection). HCG was pretreated with HEP at dosage of 0.2 g/L in drinking water [26], once daily during the whole testing period. CICG was infected with 2000 TCID50 of MDRV one time basing on the previously established MDRV natural infection model, and then treated with physiological saline solution (0.9%, w/v), once daily during the whole testing period. HPG was also pre-treated with HEP at dosage of 0.2 g/L in drinking water, once daily during the whole testing period, plus MDRV infection same with CICG. As for MDRV natural infection model, generally, forty 2-day-old healthy Muscovy ducklings were administered by the intramuscular injection with 2000 TCID50 of MDRV as artificial challenge group, three days later, twenty artificially challenged ducklings were randomly selected to join fifty normal ducklings (CICG) or fifty HEP-pretreated ducklings (HPG) with a 2:5 ratio for cohabitation infection, and the following day was referred to as 1 day post-infection (dpi) [26]. Small intestinal samples were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections and radioimmunoassay of sIgA and partial cytokines. All of the experimental Muscovy ducklings were allowed to have free access to standard complete formula feed for ducklings and drinking water during the experiment. Each group was adopted in separated cages and maintained under the identical normal conditions of temperature, humidity and light. The clinical symptoms and dead cases in each group were observed and recorded every day for further evaluation.
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2.4. Histological observation of duodenum
3. Results
For intestinal morphology analysis, about 0.5 cm duodenum tissues were collected, washed with pre-chilled phosphate buffered saline (PBS, pH 7.2) and then fixed in Bouin solution for 24 h, followed by water flush overnight. Then tissues were dehydrated in a graded ethanol series, clarified with fresh xylene, and then embedded into paraffins. Serial sections of 5 m thickness were prepared and stained with hematoxylin and eosin (HE), and then analyzed with a TESA ETALON TCM100 light microscope (Switzerland) under a magnification of 100× or 400× . For each group, five sections were selected for observation of the morphological structures of duodenum, and determination of the related parameters including intestinal villus height/crypt depth (V/C) ratio and villous surface area (VSA). Ten different regions were randomly selected in each section. The villus height was measured from the crypt opening to the tip of the villus, and the crypt depth was measured from the opening to basing of crypt. VSA = 2rH, r stands for half of the villus width, H stands for villus height.
3.1. Basic characteristics of the purified HEP
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The prepared HEP was purified and then subject to characterization of the basic characteristic. The carbohydrate content of the prepared HEP was 75.05%, the average MW of HEP determined with GPC method was approximately 16.18 kDa. The monosaccharide composition of purified HEP was determined with HPLC, and results showed that the HEP was composed of glucose (51.02%), galactose (42.24%), mannose (4.5%) and arabinose (2.2%). These results were similar to our previous study [22]. The FTIR spectra of the HEP in a wavenumber range of 4000–400 cm−1 was showed in Fig. 1, the intense broad peak at 3589 cm−1 was attributed to the hydroxyl stretching vibration, a weak absorbent band appeared at 2926 cm−1 corresponding to C H stretching vibration, the signal at 1658 cm−1 was attributed to asymmetrical stretching of the carboxylate anion group (C O), and the peaks located at 1400–1000 cm−1 corresponded to C O C stretching vibration, which indicated that the purified HEP was polysaccharides. 3.2. Changes in the morphological structures of duodenum
2.5. Immunocytes counts in duodenal endothelium The duodenums of Muscovy ducklings in each group were collected at indicated time points for determination of intraepithelial lymphocytes (IELs), goblet cells and mast cells counts. For IELs counts, tissue slides were prepared and stained with HE method. The numbers of IELs were counted in five longest intestinal villi per each of three sections per group, and expressed as number of cells per 100 columnar epithelial cells [27]. As for goblet cells and mast cells counts, tissue slides were prepared as above described, and then respectively stained with Glycogen periodic acid-Schiff (PAS) and Toluidine blue according to manufacturer’s instructions of corresponding kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The counts of five visual fields in each slice from three sections per group were counted and then average numbers of goblet cells and mast cells per view (over areas of 0.01 mm2 ) were calculated for each group [27].
2.6. Radioimmunoassay The intestinal mucosa was scraped from 5.0 cm tissues of the duodenum collected at indicated time points and weighed, and added into pre-chilled PBS solution (pH 7.2) with a weight: volume ratio of 1:2 and incubated at 4 ◦ C for 30 min. Next, tissues were sonicated for 5 min in an ultrasonic cleaner, and centrifuged for 15 min with 8000 r/min at 4 ◦ C. The resulting supernatant was collected for the determination of sIgA, IFN-␥ and IL-4 productions with radioimmunoassay kit following by the manufacturer’s instructions from Beijing Huaying Biotechnology Research Institute (Beijing, China). The examination was independently performed in triplicate.
2.7. Statistical analysis All the statistical data were presented as the means ± standard deviations (SD), and statistical analysis was performed with oneway analysis of variance (ANOVA). Least significant difference (LSD) tests were used to analyze statistical significance of differences between groups with SPSS 22.0 software. A level of P < 0.05 was considered statistically significant.
The duodenum tissues collected from experimental Muscovy ducklings at 15 dpi were prepared for tissue sections and stained with HE method. Under the optical microscope, duodenal villi were intact and closely arranged in BCG and HCG (Fig. 2A and B). However, serious lesions of duodenal villi were observed in Muscovy ducklings from CICG, including incomplete structure, mucosal shedding, villus atrophy, sparse and irregular arrangement, etc. (Fig. 2C). Compared with CICG, HEP pretreatment in HPG could efficiently improve MDRV-caused intestinal mucosal injuries, presenting longer and thicker villi, narrower villus spatium, relatively regular arrangement as well as complete structure of villi (Fig. 2D). 3.3. Changes in the intestinal parameters of duodenum The duodenal villus height/crypt depth (V/C) ratios and villous surface area (VSA) in experimental Muscovy ducklings were showed in Fig. 3A and B, respectively. The pre-treatment of HEP in HCG significantly (P < 0.05) increased the V/C ratios and VSA from 6 dpi to 21 dpi in a time-dependent manner in comparison with the BCG. As for the CICG, the V/C ratios and VSA were significantly (P < 0.05) lower than those in the BCG from 3 dpi to 21 dpi. However, pre-treatment of HEP in the MDRV-infected ducklings (HPG) significantly (P < 0.05) increased the V/C ratios and VSA from 3 dpi to 21 dpi in a time-dependent manner in comparison with the CICG. Although the V/C ratios and VSA in HPG were still significantly (P < 0.05) lower than those in BCG from 6 dpi to 21 dpi, the results obviously suggested that HEP administration not only remarkably increased the V/C ratios and VSA in normal Muscovy ducklings, but also efficiently recovered the V/C ratios and VSA in MDRV-infected Muscovy ducklings. 3.4. Changes in IELs, goblet cells and mast cells counts in the duodenal endothelium The intraepithelial lymphocytes (IELs) in the duodenal endothelium of Muscovy ducklings were stained with HE method, under the optical microscope, IELs were displayed with deep purple nuclei and were dispersed between the columnar epithelial cells of the duodenal villi, which could be found in both basal and top region of the epithelium in duodenum. In our study, MDRV infection led to the decrease of IELs at the top of the epithelial cells, and HEP treatment in the HPG obviously increased the IELs at the top of the epithelial cells when compared with the CICG (Fig. 4A). The gob-
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Fig. 1. Infrared spectra of the purified HEP in the frequency range of 400–4000 cm−1 .
let cells in the duodenal endothelium of Muscovy ducklings had goblet-like appearance and were stained with Glycogen periodic acid-Schiff (PAS) method, under the optical microscope, the goblet cells were displayed with red or purple cytoplasm, and mainly distributed at the surface layer between the columnar epithelial cells of the duodenal villi. Most goblet cells were located at the middle and top region of villi at 15 dpi. In our study, MDRV infection caused remarkable decrease of goblet cells counts in duodenal villi, as well as cellular shrink and unclear staining with light red. While HEP treatment in the infected ducklings significantly increased the goblet cells counts with normal staining in comparison with the CICG (Fig. 5A). The mast cells in the duodenal endothelium of Muscovy ducklings were stained with Toluidine blue method, under the optical microscope, the goblet cells were displayed with purple red appearance, and mainly distributed in the connective tissues of lamina propria in the centraxonial region of villi, and the junction between lamina propria and mucosal epithelium as well as surroundings of intestinal gland. In our study, MDRV infection caused remarkable decrease of goblet cells counts and cellular shrink in duodenal villi. While HEP treatment in the infected ducklings significantly increased the mast cells counts with normal shape compared with the CICG (Fig. 6A). The dynamics of IELs and goblet cells counts in the duodenal endothelium of each group were showed in Figs. 4 B and 5 B, respectively. The numbers of IELs in HCG were significantly increased (P < 0.05) from 6 dpi to 21 dpi and the goblet cells counts in HCG were significantly increased (P < 0.05) during the whole testing period (1–21 dpi) in a time-dependent manner in comparison with the BCG. When compared with BCG, MDRV infection significantly increased (P < 0.05) the IELs and goblet cells counts at 3 dpi, following by significant decrease (P < 0.05) from 6 dpi to 21 dpi. However, pre-treatment of HEP in the infected ducklings significantly inhibited the excessive elevation and following reduction of
IELs and goblet cells counts caused by MDRV infection from 3 dpi to 21 dpi in a time-dependent manner. The dynamics of mast cells counts in the duodenal endothelium of each group were showed in Fig. 6B. The numbers of mast cells in HCG displayed different degrees of increase during the whole testing period (1–21 dpi) in a time-dependent manner compared with the BCG. The mast cells count in the duodenum from 3 dpi to 21 dpi significantly (P < 0.05) decreased in MDRV-infected ducklings compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the mast cells counts from 3 dpi to 21 dpi in a time-dependent manner when compared with the CICG. Collectively, although the IELs, goblet cells and mast cells counts in HPG were still remarkably lower than those in the BCG from 6 dpi to 21 dpi, our results indicated that HEP administration not only significantly increased the duodenal immunocytes counts in normal Muscovy ducklings, but also efficiently recovered the immunocytes counts in MDRV-infected Muscovy ducklings. 3.5. Changes in the sIgA, IFN- and IL-4 contents in the duodenum Changes in the sIgA, IFN-␥ and IL-4 contents in the duodenum from each group were detected with radioimmunoassay, and the dynamics of sIgA contents in the duodenum were showed in Fig. 7A. The sIgA contents in the duodenum in HCG presented certain degrees of increase during the whole testing period (1–21 dpi) in comparison with the BCG (P > 0.05). MDRV infection significantly (P < 0.05) decreased the sIgA secretions from 1 dpi to 21 dpi compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the sIgA secretions from 1 dpi to 21 dpi compared with the CICG, and there were no significant differences between HPG and BCG (P > 0.05). Collectively, our results suggested that HEP administration not only numeri-
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Fig. 2. Effect of HEP on duodenal morphological structure in Muscovy ducklings. Duodenums from each group at 15 dpi were prepared for tissue sections and stained with HE method, and then duodenal morphological structures were analyzed with a TESA ETALON TCM100 light microscope (Switzerland) under a magnification of 100×. (A) Blank control group (BCG), treated with physiological saline solution (0.9%, w/v); (B) HEP control group (HCG), treated with 0.2 g/L HEP in drinking water; (C) cohabitation infection control group (CICG), infected with 2000 TCID50 of MDRV; (D) HEP prevention group (HPG), treated with 0.2 g/L HEP in drinking water plus MDRV infection.
Fig. 3. Effect of HEP on intestinal villus height/crypt depth (V/C) ratios (A) and villous surface area (B) in experimental Muscovy ducklings. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with HE staining, and then five sections per group were selected for determination of the V/C ratios and villous surface area. Bars without the same superscripts (a–d) mean significant differences (P < 0.05). BCG, treated with physiological saline (0.9%, w/v); HCG, treated with 0.2 g/L HEP in drinking water; CICG, infected with 2000 TCID50 of MDRV; HPG, treated with 0.2 g/L HEP in drinking water plus MDRV infection.
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Fig. 4. Effect of HEP on IELs in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with HE staining, and then three sections per group were selected for determination of the IELs distribution and counts. (A) The distribution of IELs (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic IELs counts in the duodenal endothelium from each group. The results were expressed as number of cells per 100 columnar epithelial cells, and bars without the same superscripts (a–d) mean significant differences (P < 0.05).
cally increased the duodenal sIgA secretions in normal Muscovy ducklings, but also significantly recovered the sIgA secretions in MDRV-infected Muscovy ducklings. The dynamics of IFN-␥ contents in the duodenum were showed in Fig. 7B, there were no significant differences between HCG and BCG at each time points (P > 0.05), and MDRV infection significantly (P < 0.05) suppressed the IFN-␥ productions from 3 dpi to 15 dpi compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the IFN-␥ secretions from 3 dpi to 21 dpi in comparison with the CICG, and there were no significant differences between HPG and BCG (P > 0.05) at all selected time points except an obvious decrease at 3 dpi. The dynamics of IL4 contents in the duodenum were showed in Fig. 7C, there were no significant differences between HCG and BCG (P > 0.05) at all selected time points except a significant increase at 15 dpi. The IL-4 contents in the duodenum in CICG displayed certain degrees of decrease in comparison with the BCG during the whole testing period (1–21 dpi), with a statistically significant decrease at 10 dpi. In our study, pre-treatment of HEP in the infected ducklings reversed the IL-4 decrease caused by MDRV infection, and significantly (P < 0.05) increased the IL-4 secretions at 1 dpi, 10 dpi and 21dpi when compared with the CICG.
4. Discussion Intestinal mucosa is an important immune barrier against exogenous pathogens and toxins [28], and the integrity of its morphological structure directly affects the intestinal mucosal barrier function and intestinal absorptive capacity of nutrients [29]. Intestinal mucosal immune barrier is an indispensable part of the body’s immune system and plays a great role in the fight against pathogenic microorganisms, whose functional studies have become a focus of medical research recently. Considering the anti-virus effect, anti-tumor effect and the regulation of immune response of HEP [17–20], we previously used HEP in the prevention and treatment of MDRV infection, and have found that HEP can effectively alleviate the clinical symptoms and reduce mortality in sick Muscovy ducklings [12,26]. In the present study, we first report that HEP effectively repair the injured small intestinal mucosa and restore the intestinal mucosal barrier functions in MDRV-infected Muscovy ducklings, which can be used as a potentially effective Chinese medicine preparation for clinical control of MDRV. In our study, we first found that MDRV infection caused obvious morphological changes of small intestinal mucosa in Muscovy ducklings, including the necrosis and shedding of villous epithe-
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Fig. 5. Effect of HEP on goblet cells in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with Glycogen periodic acid-Schiff (PAS) staining, and then three sections per group were selected for determination of distribution and counts of the goblet cells. (A) The distribution of goblet cells (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic goblet cells counts in the duodenal endothelium from each group. The average numbers of goblet cells per visual field (over areas of 0.01 mm2 ) were calculated for each group, and bars without the same superscripts (a–d) mean significant differences (P < 0.05).
lial cells, incompleteness of villous morphology, changes in the villus arrangement and distribution as well as the apparent parameters of villi, the increasing infiltration of intestinal submucosal inflammatory cells, and other intestinal pathological damages (data not shown). The pre-treatment of HEP by drinking administration can effectively improve MDRV-induced morphological changes of intestinal mucosa from Muscovy ducklings, and maintain relatively normal intestinal morphology to sustain the intestinal mucosal immune barrier function. Next, the morphological parameters of the small intestine mucosa were determined, such as villus height/crypt depth (V/C) ratios and villous surface area (VSA). Villus height and VSA are both important indicators of the intestinal absorptive surface, and crypt depth is closely associated with the tissue turnover [30,31]. Long villi, short crypts or high V/C ratios usually reflect a healthy intestinal system with high brush border enzyme activity [32]. In this study, we observed that MDRV infection could inhibit the growth of duodenum and significantly decrease the V/C ratio and villus surface area, leading to the injuries of innate immune barrier and the decrease in digestive enzyme activity and absorptive function of small intestine, which possibly explain the reason why MDRV infection causes a high mortality in Muscovy ducklings and the tol-
erant Muscovy ducklings usually appear growth stagnation [26]. Nevertheless, the HEP pretreatment significantly increased the V/C ratio and VSA in normal Muscovy ducklings, and efficiently recovered the above indexes in MDRV-infected Muscovy ducklings in a time-dependent manner, suggesting that HEP has the intestinal protective effect. HEP can promote the regeneration of intestinal mucosal epithelium, and restore intestinal mucosal morphology and non-specific mucosal barrier function in MDRV-infected Muscovy ducklings, which is beneficial to the recovery of this disease. The status of immune function in local intestinal mucosa is to some extent reflected by changes in the numbers of immunerelated cells. Intraepithelial lymphocytes (IELs), goblet cells and mast cells are important parts of intestinal mucosal epithelial cells, and are responsible for the maintenance of intestinal morphology and also serve as the first defense barrier of intestinal mucosal immune system against the invasion of foreign pathogens or antigens [33]. To further detect the degree of injury of intestinal mucosal immune barrier in MDRV-infected Muscovy ducklings and explore the repair mechanism of HEP in this progress, the dynamic changes in distribution and numbers of intestinal mucosal immune-related cells were measured in this study.
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Fig. 6. Effect of HEP on mast cells in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with Toluidine blue staining, and then three sections per group were selected for determination of distribution and counts of the mast cells. (A) The distribution of mast cells (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic mast cells counts in the duodenal endothelium from each group. The average numbers of mast cells per visual field (over areas of 0.01 mm2 ) were calculated for each group, and bars without the same superscripts (a–d) mean significant differences (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Intestinal IELs are a group of T lymphocytes interdigitated between epithelial cells in many mucosal sites. IELs are the front guardian of the intestinal mucosal immune system, upon encountering antigens, they immediately release cytokines (such as IL-2, IL-18 and IFN-␥) for immune defense and cause killing of infected target cells [34,35]. The number of IELs is a good indicator for evaluating the structural and functional integrity of the local mucosal immune barrier in the small intestine [36–38]. In our study, MDRV obviously inhibited the migration of IELs to the top of the epithelial cells in the late period of MDRV infection, which probably resulted in the reduction of the immune function of intestinal mucosa. HEP treatment in the infected ducklings significantly increased the IELs at the top of the epithelial cells compared with the CICG, which indicated that HEP could promote the maturation of IELs and migration of the matured IELs into the intestinal tract to react with the antigens for maintenance of intestinal mucosal immune function [39]. Goblet cells are also important immunocytes and are able to secrete intestinal mucus that serves as a diffuse barrier protecting the intestinal mucosal epithelium [31]. The formed mucus layer contains a large number of secretory immunoglobulin (sIgA), which is the major antibody in digestive liquids, and combines with pathogenic microorganisms and then prevents them from adhering
to mucosal epithelium [40]. Goblet cells also synthesize and secrete intestinal trefoil factor (TFF3), which is essential in maintaining mucosal integrity in gastrointestinal tract. Our results showed that MDRV caused remarkable decrease of goblet cells at the upper region of the duodenal villi, as well as cellular shrink and unclear staining with light red in the late period of MDRV infection. While HEP treatment in the infected ducklings significantly increased the goblet cells counts with normal staining at the upper region of the duodenal villi, which could efficiently maintain the secretion of intestinal mucus and mucosal integrity in intestinal tract, and then promote the repair of the injured intestinal mucosa. MDRV infection induced a significant elevated IELs and goblet cells numbers at 1 dpi–3 dpi in comparison with the blank control, which might be related to the fight against intestinal mucosal inflammation. While the two indexes significantly decreased at 6 dpi–21 dpi in MDRVinfected Muscovy ducklings, it might result from the apoptosis or necrosis of intestinal intraepithelial lymphocytes and the impairment of intestinal mucosal barrier [21,41]. However, pre-treatment of HEP in the infected ducklings could efficiently inhibit the excessive up-regulation and following reduction of IELs and goblet cells counts induced by MDRV infection from 3 dpi to 21 dpi in a timedependent manner. It was possibly associated with the reduction of
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Fig. 7. Effect of HEP on duodenal sIgA, IFN-␥ and IL-4 contents in experimental Muscovy ducklings. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for determination of the dynamic sIgA, IFN-␥ and IL-4 secretions using radioimmunoassay. The exam was performed in triplicate. Bars without the same superscripts (a–d) mean significant differences (P < 0.05).
inflammation at early stage of MDRV infection and the suppression of IELs and goblet cells apoptosis at late infection. Mast cells are not only the effector cells in native immunity, but also participate in acquired immunity, which are the firstline cells for anti-infection immunity. Upon contacting with the foreign antigens, mast cells can quickly and selectively produce active mediators to trigger protective immune responses [42]. And evidences have revealed that functional enhancement of mast cells can up-regulate the body’s anti-virus ability [43]. Our results showed that late infection of MDRV caused remarkable decrease of mast cells counts and cellular shrink in lamina propria of duodenal mucosa, which suggested that MDRV inhibited the growth and development of mast cells, and brought damages to intestinal native and acquired immune response. While HEP treatment in the infected ducklings significantly increased the mast cells counts with normal shape, therefore, HEP can promote the development of mast cells in small intestine to ensure the anti-virus ability of Muscovy ducklings. Collectively, HEP pretreatment not only downregulates the numbers of IELs and goblet cells in the early stage of MDRV infection to reduce the inflammation of the body, but also promote the recovery of IELs, goblet cells and mast cells in the late stage of infection to promote the repair of the intestinal mucosal immune barrier in MDRV-infected Muscovy ducklings. Mucosal immunity is the first line in the fight of body against the enteroviruses and respiroviruses. Plenty of sIgA is produced by goblet cells in the gastrointestinal tract and is the main effector in
mucosal immune response, when the viruses invade the body, they stimulate the body to secrete a large number of sIgA and then sIgA combines with the viruses adhering to mucosal surface to prevent the virus from invading epithelial cells, simultaneously plentiful mucus proteins are produced to kill the virus [44]. In our study, we also detected the effect of HEP on intestinal sIgA levels, and the results showed that the sIgA secretions in intestinal mucosa in MDRV-infected Muscovy ducklings were significantly lower than those in any of the groups, indicating that MDRV impaired the intestinal mucosal immune function in Muscovy ducklings, which is potentially associated with the destruction of goblet cells. The HEP administration in drinking water significantly increased the intestinal sIgA secretion, thus enhancing intestinal mucosal immune function and reducing MDRV-induced damages to Muscovy ducklings. Additionally, dynamic IFN-␥ and IL-4 secretions were detected in our study. IFN-␥, or type II IFN, is a representative of Th1 cytokines and plays a great role in the native and acquired immunity against viral and intracellular bacterial infections [45]. IL-4 is a kind of Th2 cytokines and can promote the proliferation and differentiation of T lymphocytes, B lymphocytes and mast cells, and also plays an important role in the formation of sIgA [46]. IL-4 has been shown to downregulate the inflammatory response through inhibition of IL-8 [47]. In the intestinal epithelia, IELs are the major source of IFN-␥, in addition, both IELs and mast cells can produce IL-4 to perform immunoregulation. Our results revealed that MDRV infection significantly suppressed the IFN-␥
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secretions at the early stage of viral infection (3–6 dpi), from 6 dpi till to 21 dpi, IFN-␥ contents in CICG gradually increased but were still lower than those in BCG. Oral administeration of HEP in the infected ducklings significantly (P < 0.05) increased the IFN-␥ secretions in comparison with the CICG, which is beneficial to recovery or enhancement of immune response in sick ducklings. Our results also showed that duodenal IL-4 contents in CICG displayed certain degrees of decrease in comparison with the BCG during the whole testing period (1–21 dpi), and pre-treatment of HEP in the infected ducklings could reverse the IL-4 decrease caused by MDRV infection and maintain the dynamic balance of IL-4 levels, which is helpful to relieve inflammation and injures of intestinal mucosal immune functions. In conclusion, our findings indicate that HEP pre-treatment can significantly improve the intestinal mucosal morphology and related parameters, the numbers of intestinal mucosal immunityrelated immunocytes as well as intestinal sIgA, IFN-␥ and IL-4 secretion in MDRV-infected Muscovy ducklings, thus repairing injures of small intestinal mucosal immune barrier, and improving immune responses and reducing the mortality rate of sick Muscovy ducklings. HEP can be used as a potential natural Chinese medicine extract for clinical prevention and control of MDRV infection and other animal diseases. In addition, we also made a preliminary exploration on the molecular mechanism of HEP in repairing the intestinal mucosa (data not shown). Our findings will provide valuable reference for clarification of the protective mechanism of HEP on intestinal mucosa and further application of HEP in control of clinical diseases. Acknowledgments This research was supported by grants from the Natural Science Foundation of China (Grant Number: 31372474) and grants from the Natural Science Foundation of Fujian Province (Grant Number: 2017J01597) References [1] V.R. Kaschula, A new virus disease of the Muscovy-duck [Cairina moschata (linn)] present in Natal, J. S. Afr. Vet. Assoc. 21 (1950) 18–26. [2] D. Gaudry, J.M. Charles, J. Tektoff, A new disease expressing itself by a viral pericarditis in Barbary ducks, C. R. Acad. Sci. Hebd. Seances Acad. Sci. D 274 (1972) 2916–2919. [3] M. Malkinson, K. Perk, Y. Weisman, Reovirus infection of young Muscovy ducks (Cairina moschata), Avian Pathol. 10 (1981) 433–440. [4] U. Heffels-Redmann, H. Muller, E.F. Kaleta, Structural and biological characteristics of reoviruses isolated from Muscovy ducks (Cairina moschata), Avian Pathol. 21 (1992) 481–491. [5] B.C. Wu, J.X. Chen, J.S. Yao, Z.H. Chen, W.L. Chen, G.P. Li, X.C. Zeng, Isolation and identification of Muscovy duck reovirus, J. Fujian Agric. Univ. 30 (2001) 227–230. [6] G. Kuntz-Simon, G. Le Gall-Reculé, C. de Boisséson, V. Jestin, Muscovy duck reovirus sigmaC protein is atypically encoded by the smallest genome segment, J. Gen. Virol. 83 (2002) 1189–1200. [7] B.C. Wu, J.X. Chen, J.S. Yao, Pathogenicity of Muscovy duck reovirus isolate B3, Chin. J. Prev. Vet. Med. 23 (2001) 422–425. [8] B.C. Wu, J.S. Yao, J.X. Chen, H.M. Lu, Z.H. Chen, Pathology of infection with reovirus isolate B3 in Muscovy ducks, J. Fujian Agric. Univ. 30 (2001) 514–517. [9] S.Y. Chen, Q.L. Hu, X.X. Cheng, S.L. Chen, B. Jiang, T.L. Lin, F.Q. Lin, X.L. Zhu, Y.Q. Cheng, Ultrastructure observation of muscovy ducks infected with Muscovy duck reovirus, Chin. J. Vet. Sci. 26 (2006) 662–664. [10] Q.X. Wang, L.J. Zhao, F. Su, B.C. Wu, G.P. Li, Effects of infection with Muscovy duck reovirus isolate B3 on the cellular immunity function of Muscovy duck, J. Fujian Agric. Univ. 38 (2009) 380–383. [11] J.X. Jiang, L.N. Huang, H.H. Jiang, J. Fang, D.B. Li, X. Cheng, Y.J. Wu, B.C. Wu, Effect of Astragalus polysaccharides on levels of cytocines in Muscovy ducks infected with Muscovy duck reovirus, Chin. Vet. Sci. 45 (2015) 873–880. [12] L.H. Zheng, Y.J. Wu, J.L. Liao, D.L. Cai, Y.Y. Zhang, E.P. Zhu, W.D. Zhou, B.C. Wu, Y.F. Huang, Chin. Vet. Sci. 2 (2017) 249–256. [13] G. Kuntzsimon, P. Blanchard, M. Cherbonnel, A. Jestin, V. Jestin, Baculovirus-expressed Muscovy duck reovirus sigmaC protein induces serum neutralizing antibodies and protection against challenge, Vaccine 20 (2002) 3113–3122.
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Hericium erinaceus polysaccharide facilitates restoration of injured intestinal mucosal immunity in Muscovy duck reovirus-infected Muscovy ducklings Yijian Wu a,b , Huihui Jiang a,c , Erpeng Zhu a,b , Jian Li a,b , Quanxi Wang a,b , Wuduo Zhou a,c , Tao Qin a,b , Xiaoping Wu a,c , Baocheng Wu a,b , Yifan Huang a,b,∗ a
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, People’s Republic of China Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou 350002, People’s Republic of China c University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fuzhou 350002, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 14 June 2017 Received in revised form 13 September 2017 Accepted 22 September 2017 Available online xxx Keywords: Muscovy duck reovirus Hericium erinaceus polysaccharide Duodenum Mucosal immunity Restoration
a b s t r a c t To elucidate the effect of Hericium erinaceus polysaccharide (HEP) on the intestinal mucosal immunity in normal and Muscovy duck reovirus (MDRV)-infected Muscovy ducklings, 1-day-old healthy Muscovy ducklings were pretreated with 0.2 g/L HEP and/or following by MDRV infection in this study, duodenal samples were respectively collected at 1, 3, 6, 10, 15 and 21 day post-infection, tissue sections were prepared for observation of morphological structure and determination of intestinal parameters (villus height/crypt depth ratio, villus surface area) as well as counts of intraepithelial lymphocytes (IELs), goblet cells, mast cells. Additionally, dynamics of secretory immunoglobin A (sIgA), interferon-␥ (IFN-␥) and interleukin-4 (IL-4) productions in intestinal mucosa were measured with radioimmunoassay. Results showed that HEP significantly improved intestinal morphological structure and related indexes, and significantly inhibited the reduction of intestinal mucosal IELs, goblet cells and mast cells caused by MDRV infection. Furthermore, HEP significantly increased the secretion of sIgA, IFN-␥ and IL-4 to enhance intestinal mucosal immune functions. Our findings indicate that HEP treatment can effectively repair MDRV-caused injures of small intestinal mucosal immune barrier, and improve mucosal immune function in sick Muscovy ducklings, which will provide valuable help for further application of HEP in prevention and treatment of MDRV infection. © 2017 Published by Elsevier B.V.
1. Introduction Muscovy duck reovirus (MDRV) causes an infectious disease in Muscovy ducklings with a high morbidity and mortality. Acute cases of MDRV infections were first described in South Africa in 1950 [1], and the virus was isolated from sick ducks in France in 1972 [2], and later in Israel, Germany and other regions [3,4]. In China, MDRV infection first appeared in 1997, and usually named as “liver white spot disease”, which is characterized clinically by soft foot, diarrhea, stunted growth and white necrotic foci in liver and spleen [2,5,6], resulting in huge economic losses in Muscovy
∗ Corresponding author at: Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health (Fujian Agricultural and Forestry University), Fuzhou 350002, People’s Republic of China. E-mail address: [email protected] (Y. Huang).
duck industry. The etiological agent of “liver white spot disease” was isolated and first defined as MDRV by our group in 2001 [5], which belongs to the family Reoviridae, genus Orthoreovirus. Further studies have showed that MDRV infection induces serious immunosuppression in sick Muscovy ducklings, including severe damages to central immune organs (bursa of Fabricius, thymus), peripheral immune organs (spleen) as well as the associated cellular and humoral immunoresponses [7–10]. Reduction of the immune protections from vaccines and other pathogens secondary to MDRV infection often aggravate this disease due to the suppression of immune function in sick Muscovy ducklings. Intestinal mucosal immunity in animals especially in poultry plays an important part in the body’s immune system. We have previously found that MDRV infection causes serious damage to intestinal mucosa and intestinal mucosal immune system, as well as absorption disorders in Muscovy ducklings [11,12], thus exacerbating the development and outcome of this disease. Further studies have showed that
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MDRV-caused mucosal immunologic injuries are probably associated with intestinal mucosal inflammation, bleeding, degeneration, as well as necrosis, apoptosis and large decreases of lymphocytes in mucosa-associated immune tissues (cecal tonsils) [7,8]. As a kind of immunosuppressive disease with high mortality rate, MDRV infection has attracted great attentions of researchers. Since the genome of MDRV consists of ten segments of doublestranded RNA (dsRNA) and is prone to genetic variation and recombination, the diversifications of disease patterns as well as expansion of MDRV infection spectrum and epidemic areas generally occur, thus greatly increasing the difficulties of prevention and control for MDRV. Domestic researchers have developed MDRV attenuated vaccine, inactivated vaccine and genetic engineering vaccine to prevent the disease [13–15], however, whether the vaccines are able to provide enough safe protection remains to be further verified. Currently, there are no commercially available agents for treatment of MDRV-induced immunosuppression. During the course of developing efficient approaches to deal with immunosuppression caused by MDRV, our group has performed long-term researches. Many traditional Chinese medicine polysaccharide extracts have immunomodulatory and immune-enhancing functions with no drug resistance and residue problems, from which to find potential candidates to improve or cure MDRV-caused immunosuppressive injuries will be of great significance. Hericium erinaceus polysaccharide (HEP) is a kind of polysaccharide extracts from H. erinacues fruiting body, mycelia and mycelial fermentation broth, which is naturally non-toxic and is one of the most important active substances in H. erinacues [16]. HEP has many biological activities, such as enhancing immune functions, scavenging free radicals, as well as facilitating regeneration and repair of gastrointestinal mucosa [17–20]. Considering these advantages, we have previously exploited HEP to treat MDRV infection and have found that HEP pre-treatment could efficiently protect Muscovy ducklings from MDRV infection, including alleviating clinical symptoms, reducing morbidity and mortality and promoting recovery of sick Muscovy ducklings [12,21]. However, the main mechanism remains unclear and needs to be further studied. To elucidate the effect of HEP on the intestinal mucosal immunity in MDRV-infected Muscovy ducklings, in the present study, healthy Muscovy ducklings were pretreated with HEP and then infected with MDRV based on the established MDRV natural infection model, and then morphological structure and related parameters of small intestinal mucosa, counts of mucosal immunocytes such as intraepithelial lymphocytes (IELs), goblet cells and mast cells, as well as sIgA, Th1 cytokine (IFN-␥) and Th2 cytokine (IL-4) productions in intestinal tissues were determined. This study will provide valuable help for elucidating the immunomodulatory mechanism of HEP on intestinal mucosal immunosuppression, and for further application of HEP in prevention and treatment of MDRV infection.
2. Materials and methods 2.1. Materials and chemicals H. erinacues were purchased from Huishihao Pharmacy in Fujian Province of China. Muscovy duck reovirus (MDRV) YB strain was isolated and stored by our laboratory [5]. Slides, Toluidine blue staining kit and Glycogen periodic acid-Schiff (PAS) staining kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The radioactive immunoassay kit for secretory immunoglobulin A (sIgA), interferon-␥ (IFN-␥) and interleukin4 (IL-4) were purchased from Beijing Huaying Biotechnology Research Institute (Beijing, China). All the other chemicals used in this study were of analytical grade.
2.2. Preparation and characterization of H. erinaceus polysaccharides The H. erinaceus polysaccharides (HEP) were extracted from fruiting bodies of H. erinaceus according to the procedures described in our previous study [22]. Briefly, 1000 g of dried H. erinaceus was crushed into small pieces, soaked with 95% ethanol, and refluxed twice in water bath of 80 ◦ C. After drying, the drug was decocted with 20-fold volume water 3 times each for 30 min. The mixture was filtrated through two-layer gauze, and the retentate was concentrated into 1000 mL and then mixed with 95% (v/v) ethanol to yield a 90% ethanolic solution. Finally, the precipitation was lyophilized to obtain the crude HEP with a vacuum freezedrying machine (Model LGJ-25, Dongxing Machinery Industry Co., Ltd. Shamen City). Proteins in the crude HEP were eliminated with Sevage method [23], and the products were further purified by DEAE-52 cellulose and Sephadex G-100 (1.5 cm × 200 cm). The obtained main polysaccharides fraction was subjected to determination of HEP content by the phenol-sulfuric acid method [24,25], and then freeze-dried to get the purified HEP, which was then used in our following biological studies. Besides, as previously described, the purified HEP was subject to Fourier-transform infrared spectroscopy (FTIR) analysis, and the average molecular weight (MW ) of the HEP was measured through gel permeation chromatography (GPC) method, and the monosaccharide composition of the HEP was determined by high-performance lipid chromatography (HPLC) [17,19,22].
2.3. Animals and experimental treatments Two hundred 1-day-old healthy Muscovy ducklings weighing 44.85 ± 9.23 g were provided by the Putian (Guangdong) Wen’s poultry Co., Ltd (Fujian, China), and randomly divided into four groups, blank control group (BCG), HEP control group (HCG), cohabitation infection control group (CICG) and HEP prevention group (HPG), each consisting of 50 ducklings. BCG was only treated with physiological saline solution (0.9%, w/v), once daily during the whole testing period (till to 21-day post-infection). HCG was pretreated with HEP at dosage of 0.2 g/L in drinking water [26], once daily during the whole testing period. CICG was infected with 2000 TCID50 of MDRV one time basing on the previously established MDRV natural infection model, and then treated with physiological saline solution (0.9%, w/v), once daily during the whole testing period. HPG was also pre-treated with HEP at dosage of 0.2 g/L in drinking water, once daily during the whole testing period, plus MDRV infection same with CICG. As for MDRV natural infection model, generally, forty 2-day-old healthy Muscovy ducklings were administered by the intramuscular injection with 2000 TCID50 of MDRV as artificial challenge group, three days later, twenty artificially challenged ducklings were randomly selected to join fifty normal ducklings (CICG) or fifty HEP-pretreated ducklings (HPG) with a 2:5 ratio for cohabitation infection, and the following day was referred to as 1 day post-infection (dpi) [26]. Small intestinal samples were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections and radioimmunoassay of sIgA and partial cytokines. All of the experimental Muscovy ducklings were allowed to have free access to standard complete formula feed for ducklings and drinking water during the experiment. Each group was adopted in separated cages and maintained under the identical normal conditions of temperature, humidity and light. The clinical symptoms and dead cases in each group were observed and recorded every day for further evaluation.
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2.4. Histological observation of duodenum
3. Results
For intestinal morphology analysis, about 0.5 cm duodenum tissues were collected, washed with pre-chilled phosphate buffered saline (PBS, pH 7.2) and then fixed in Bouin solution for 24 h, followed by water flush overnight. Then tissues were dehydrated in a graded ethanol series, clarified with fresh xylene, and then embedded into paraffins. Serial sections of 5 m thickness were prepared and stained with hematoxylin and eosin (HE), and then analyzed with a TESA ETALON TCM100 light microscope (Switzerland) under a magnification of 100× or 400× . For each group, five sections were selected for observation of the morphological structures of duodenum, and determination of the related parameters including intestinal villus height/crypt depth (V/C) ratio and villous surface area (VSA). Ten different regions were randomly selected in each section. The villus height was measured from the crypt opening to the tip of the villus, and the crypt depth was measured from the opening to basing of crypt. VSA = 2rH, r stands for half of the villus width, H stands for villus height.
3.1. Basic characteristics of the purified HEP
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The prepared HEP was purified and then subject to characterization of the basic characteristic. The carbohydrate content of the prepared HEP was 75.05%, the average MW of HEP determined with GPC method was approximately 16.18 kDa. The monosaccharide composition of purified HEP was determined with HPLC, and results showed that the HEP was composed of glucose (51.02%), galactose (42.24%), mannose (4.5%) and arabinose (2.2%). These results were similar to our previous study [22]. The FTIR spectra of the HEP in a wavenumber range of 4000–400 cm−1 was showed in Fig. 1, the intense broad peak at 3589 cm−1 was attributed to the hydroxyl stretching vibration, a weak absorbent band appeared at 2926 cm−1 corresponding to C H stretching vibration, the signal at 1658 cm−1 was attributed to asymmetrical stretching of the carboxylate anion group (C O), and the peaks located at 1400–1000 cm−1 corresponded to C O C stretching vibration, which indicated that the purified HEP was polysaccharides. 3.2. Changes in the morphological structures of duodenum
2.5. Immunocytes counts in duodenal endothelium The duodenums of Muscovy ducklings in each group were collected at indicated time points for determination of intraepithelial lymphocytes (IELs), goblet cells and mast cells counts. For IELs counts, tissue slides were prepared and stained with HE method. The numbers of IELs were counted in five longest intestinal villi per each of three sections per group, and expressed as number of cells per 100 columnar epithelial cells [27]. As for goblet cells and mast cells counts, tissue slides were prepared as above described, and then respectively stained with Glycogen periodic acid-Schiff (PAS) and Toluidine blue according to manufacturer’s instructions of corresponding kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The counts of five visual fields in each slice from three sections per group were counted and then average numbers of goblet cells and mast cells per view (over areas of 0.01 mm2 ) were calculated for each group [27].
2.6. Radioimmunoassay The intestinal mucosa was scraped from 5.0 cm tissues of the duodenum collected at indicated time points and weighed, and added into pre-chilled PBS solution (pH 7.2) with a weight: volume ratio of 1:2 and incubated at 4 ◦ C for 30 min. Next, tissues were sonicated for 5 min in an ultrasonic cleaner, and centrifuged for 15 min with 8000 r/min at 4 ◦ C. The resulting supernatant was collected for the determination of sIgA, IFN-␥ and IL-4 productions with radioimmunoassay kit following by the manufacturer’s instructions from Beijing Huaying Biotechnology Research Institute (Beijing, China). The examination was independently performed in triplicate.
2.7. Statistical analysis All the statistical data were presented as the means ± standard deviations (SD), and statistical analysis was performed with oneway analysis of variance (ANOVA). Least significant difference (LSD) tests were used to analyze statistical significance of differences between groups with SPSS 22.0 software. A level of P < 0.05 was considered statistically significant.
The duodenum tissues collected from experimental Muscovy ducklings at 15 dpi were prepared for tissue sections and stained with HE method. Under the optical microscope, duodenal villi were intact and closely arranged in BCG and HCG (Fig. 2A and B). However, serious lesions of duodenal villi were observed in Muscovy ducklings from CICG, including incomplete structure, mucosal shedding, villus atrophy, sparse and irregular arrangement, etc. (Fig. 2C). Compared with CICG, HEP pretreatment in HPG could efficiently improve MDRV-caused intestinal mucosal injuries, presenting longer and thicker villi, narrower villus spatium, relatively regular arrangement as well as complete structure of villi (Fig. 2D). 3.3. Changes in the intestinal parameters of duodenum The duodenal villus height/crypt depth (V/C) ratios and villous surface area (VSA) in experimental Muscovy ducklings were showed in Fig. 3A and B, respectively. The pre-treatment of HEP in HCG significantly (P < 0.05) increased the V/C ratios and VSA from 6 dpi to 21 dpi in a time-dependent manner in comparison with the BCG. As for the CICG, the V/C ratios and VSA were significantly (P < 0.05) lower than those in the BCG from 3 dpi to 21 dpi. However, pre-treatment of HEP in the MDRV-infected ducklings (HPG) significantly (P < 0.05) increased the V/C ratios and VSA from 3 dpi to 21 dpi in a time-dependent manner in comparison with the CICG. Although the V/C ratios and VSA in HPG were still significantly (P < 0.05) lower than those in BCG from 6 dpi to 21 dpi, the results obviously suggested that HEP administration not only remarkably increased the V/C ratios and VSA in normal Muscovy ducklings, but also efficiently recovered the V/C ratios and VSA in MDRV-infected Muscovy ducklings. 3.4. Changes in IELs, goblet cells and mast cells counts in the duodenal endothelium The intraepithelial lymphocytes (IELs) in the duodenal endothelium of Muscovy ducklings were stained with HE method, under the optical microscope, IELs were displayed with deep purple nuclei and were dispersed between the columnar epithelial cells of the duodenal villi, which could be found in both basal and top region of the epithelium in duodenum. In our study, MDRV infection led to the decrease of IELs at the top of the epithelial cells, and HEP treatment in the HPG obviously increased the IELs at the top of the epithelial cells when compared with the CICG (Fig. 4A). The gob-
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Fig. 1. Infrared spectra of the purified HEP in the frequency range of 400–4000 cm−1 .
let cells in the duodenal endothelium of Muscovy ducklings had goblet-like appearance and were stained with Glycogen periodic acid-Schiff (PAS) method, under the optical microscope, the goblet cells were displayed with red or purple cytoplasm, and mainly distributed at the surface layer between the columnar epithelial cells of the duodenal villi. Most goblet cells were located at the middle and top region of villi at 15 dpi. In our study, MDRV infection caused remarkable decrease of goblet cells counts in duodenal villi, as well as cellular shrink and unclear staining with light red. While HEP treatment in the infected ducklings significantly increased the goblet cells counts with normal staining in comparison with the CICG (Fig. 5A). The mast cells in the duodenal endothelium of Muscovy ducklings were stained with Toluidine blue method, under the optical microscope, the goblet cells were displayed with purple red appearance, and mainly distributed in the connective tissues of lamina propria in the centraxonial region of villi, and the junction between lamina propria and mucosal epithelium as well as surroundings of intestinal gland. In our study, MDRV infection caused remarkable decrease of goblet cells counts and cellular shrink in duodenal villi. While HEP treatment in the infected ducklings significantly increased the mast cells counts with normal shape compared with the CICG (Fig. 6A). The dynamics of IELs and goblet cells counts in the duodenal endothelium of each group were showed in Figs. 4 B and 5 B, respectively. The numbers of IELs in HCG were significantly increased (P < 0.05) from 6 dpi to 21 dpi and the goblet cells counts in HCG were significantly increased (P < 0.05) during the whole testing period (1–21 dpi) in a time-dependent manner in comparison with the BCG. When compared with BCG, MDRV infection significantly increased (P < 0.05) the IELs and goblet cells counts at 3 dpi, following by significant decrease (P < 0.05) from 6 dpi to 21 dpi. However, pre-treatment of HEP in the infected ducklings significantly inhibited the excessive elevation and following reduction of
IELs and goblet cells counts caused by MDRV infection from 3 dpi to 21 dpi in a time-dependent manner. The dynamics of mast cells counts in the duodenal endothelium of each group were showed in Fig. 6B. The numbers of mast cells in HCG displayed different degrees of increase during the whole testing period (1–21 dpi) in a time-dependent manner compared with the BCG. The mast cells count in the duodenum from 3 dpi to 21 dpi significantly (P < 0.05) decreased in MDRV-infected ducklings compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the mast cells counts from 3 dpi to 21 dpi in a time-dependent manner when compared with the CICG. Collectively, although the IELs, goblet cells and mast cells counts in HPG were still remarkably lower than those in the BCG from 6 dpi to 21 dpi, our results indicated that HEP administration not only significantly increased the duodenal immunocytes counts in normal Muscovy ducklings, but also efficiently recovered the immunocytes counts in MDRV-infected Muscovy ducklings. 3.5. Changes in the sIgA, IFN- and IL-4 contents in the duodenum Changes in the sIgA, IFN-␥ and IL-4 contents in the duodenum from each group were detected with radioimmunoassay, and the dynamics of sIgA contents in the duodenum were showed in Fig. 7A. The sIgA contents in the duodenum in HCG presented certain degrees of increase during the whole testing period (1–21 dpi) in comparison with the BCG (P > 0.05). MDRV infection significantly (P < 0.05) decreased the sIgA secretions from 1 dpi to 21 dpi compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the sIgA secretions from 1 dpi to 21 dpi compared with the CICG, and there were no significant differences between HPG and BCG (P > 0.05). Collectively, our results suggested that HEP administration not only numeri-
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Fig. 2. Effect of HEP on duodenal morphological structure in Muscovy ducklings. Duodenums from each group at 15 dpi were prepared for tissue sections and stained with HE method, and then duodenal morphological structures were analyzed with a TESA ETALON TCM100 light microscope (Switzerland) under a magnification of 100×. (A) Blank control group (BCG), treated with physiological saline solution (0.9%, w/v); (B) HEP control group (HCG), treated with 0.2 g/L HEP in drinking water; (C) cohabitation infection control group (CICG), infected with 2000 TCID50 of MDRV; (D) HEP prevention group (HPG), treated with 0.2 g/L HEP in drinking water plus MDRV infection.
Fig. 3. Effect of HEP on intestinal villus height/crypt depth (V/C) ratios (A) and villous surface area (B) in experimental Muscovy ducklings. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with HE staining, and then five sections per group were selected for determination of the V/C ratios and villous surface area. Bars without the same superscripts (a–d) mean significant differences (P < 0.05). BCG, treated with physiological saline (0.9%, w/v); HCG, treated with 0.2 g/L HEP in drinking water; CICG, infected with 2000 TCID50 of MDRV; HPG, treated with 0.2 g/L HEP in drinking water plus MDRV infection.
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Fig. 4. Effect of HEP on IELs in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with HE staining, and then three sections per group were selected for determination of the IELs distribution and counts. (A) The distribution of IELs (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic IELs counts in the duodenal endothelium from each group. The results were expressed as number of cells per 100 columnar epithelial cells, and bars without the same superscripts (a–d) mean significant differences (P < 0.05).
cally increased the duodenal sIgA secretions in normal Muscovy ducklings, but also significantly recovered the sIgA secretions in MDRV-infected Muscovy ducklings. The dynamics of IFN-␥ contents in the duodenum were showed in Fig. 7B, there were no significant differences between HCG and BCG at each time points (P > 0.05), and MDRV infection significantly (P < 0.05) suppressed the IFN-␥ productions from 3 dpi to 15 dpi compared with the BCG. However, pre-treatment of HEP in the infected ducklings significantly (P < 0.05) increased the IFN-␥ secretions from 3 dpi to 21 dpi in comparison with the CICG, and there were no significant differences between HPG and BCG (P > 0.05) at all selected time points except an obvious decrease at 3 dpi. The dynamics of IL4 contents in the duodenum were showed in Fig. 7C, there were no significant differences between HCG and BCG (P > 0.05) at all selected time points except a significant increase at 15 dpi. The IL-4 contents in the duodenum in CICG displayed certain degrees of decrease in comparison with the BCG during the whole testing period (1–21 dpi), with a statistically significant decrease at 10 dpi. In our study, pre-treatment of HEP in the infected ducklings reversed the IL-4 decrease caused by MDRV infection, and significantly (P < 0.05) increased the IL-4 secretions at 1 dpi, 10 dpi and 21dpi when compared with the CICG.
4. Discussion Intestinal mucosa is an important immune barrier against exogenous pathogens and toxins [28], and the integrity of its morphological structure directly affects the intestinal mucosal barrier function and intestinal absorptive capacity of nutrients [29]. Intestinal mucosal immune barrier is an indispensable part of the body’s immune system and plays a great role in the fight against pathogenic microorganisms, whose functional studies have become a focus of medical research recently. Considering the anti-virus effect, anti-tumor effect and the regulation of immune response of HEP [17–20], we previously used HEP in the prevention and treatment of MDRV infection, and have found that HEP can effectively alleviate the clinical symptoms and reduce mortality in sick Muscovy ducklings [12,26]. In the present study, we first report that HEP effectively repair the injured small intestinal mucosa and restore the intestinal mucosal barrier functions in MDRV-infected Muscovy ducklings, which can be used as a potentially effective Chinese medicine preparation for clinical control of MDRV. In our study, we first found that MDRV infection caused obvious morphological changes of small intestinal mucosa in Muscovy ducklings, including the necrosis and shedding of villous epithe-
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Fig. 5. Effect of HEP on goblet cells in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with Glycogen periodic acid-Schiff (PAS) staining, and then three sections per group were selected for determination of distribution and counts of the goblet cells. (A) The distribution of goblet cells (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic goblet cells counts in the duodenal endothelium from each group. The average numbers of goblet cells per visual field (over areas of 0.01 mm2 ) were calculated for each group, and bars without the same superscripts (a–d) mean significant differences (P < 0.05).
lial cells, incompleteness of villous morphology, changes in the villus arrangement and distribution as well as the apparent parameters of villi, the increasing infiltration of intestinal submucosal inflammatory cells, and other intestinal pathological damages (data not shown). The pre-treatment of HEP by drinking administration can effectively improve MDRV-induced morphological changes of intestinal mucosa from Muscovy ducklings, and maintain relatively normal intestinal morphology to sustain the intestinal mucosal immune barrier function. Next, the morphological parameters of the small intestine mucosa were determined, such as villus height/crypt depth (V/C) ratios and villous surface area (VSA). Villus height and VSA are both important indicators of the intestinal absorptive surface, and crypt depth is closely associated with the tissue turnover [30,31]. Long villi, short crypts or high V/C ratios usually reflect a healthy intestinal system with high brush border enzyme activity [32]. In this study, we observed that MDRV infection could inhibit the growth of duodenum and significantly decrease the V/C ratio and villus surface area, leading to the injuries of innate immune barrier and the decrease in digestive enzyme activity and absorptive function of small intestine, which possibly explain the reason why MDRV infection causes a high mortality in Muscovy ducklings and the tol-
erant Muscovy ducklings usually appear growth stagnation [26]. Nevertheless, the HEP pretreatment significantly increased the V/C ratio and VSA in normal Muscovy ducklings, and efficiently recovered the above indexes in MDRV-infected Muscovy ducklings in a time-dependent manner, suggesting that HEP has the intestinal protective effect. HEP can promote the regeneration of intestinal mucosal epithelium, and restore intestinal mucosal morphology and non-specific mucosal barrier function in MDRV-infected Muscovy ducklings, which is beneficial to the recovery of this disease. The status of immune function in local intestinal mucosa is to some extent reflected by changes in the numbers of immunerelated cells. Intraepithelial lymphocytes (IELs), goblet cells and mast cells are important parts of intestinal mucosal epithelial cells, and are responsible for the maintenance of intestinal morphology and also serve as the first defense barrier of intestinal mucosal immune system against the invasion of foreign pathogens or antigens [33]. To further detect the degree of injury of intestinal mucosal immune barrier in MDRV-infected Muscovy ducklings and explore the repair mechanism of HEP in this progress, the dynamic changes in distribution and numbers of intestinal mucosal immune-related cells were measured in this study.
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Fig. 6. Effect of HEP on mast cells in the duodenal endothelium. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for preparation of tissue sections with Toluidine blue staining, and then three sections per group were selected for determination of distribution and counts of the mast cells. (A) The distribution of mast cells (black arrows) in the duodenal endothelium from each group (magnification: 400×). a–d refer to the BCG, HCG, CICG and HPG, respectively. (B) The dynamic mast cells counts in the duodenal endothelium from each group. The average numbers of mast cells per visual field (over areas of 0.01 mm2 ) were calculated for each group, and bars without the same superscripts (a–d) mean significant differences (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Intestinal IELs are a group of T lymphocytes interdigitated between epithelial cells in many mucosal sites. IELs are the front guardian of the intestinal mucosal immune system, upon encountering antigens, they immediately release cytokines (such as IL-2, IL-18 and IFN-␥) for immune defense and cause killing of infected target cells [34,35]. The number of IELs is a good indicator for evaluating the structural and functional integrity of the local mucosal immune barrier in the small intestine [36–38]. In our study, MDRV obviously inhibited the migration of IELs to the top of the epithelial cells in the late period of MDRV infection, which probably resulted in the reduction of the immune function of intestinal mucosa. HEP treatment in the infected ducklings significantly increased the IELs at the top of the epithelial cells compared with the CICG, which indicated that HEP could promote the maturation of IELs and migration of the matured IELs into the intestinal tract to react with the antigens for maintenance of intestinal mucosal immune function [39]. Goblet cells are also important immunocytes and are able to secrete intestinal mucus that serves as a diffuse barrier protecting the intestinal mucosal epithelium [31]. The formed mucus layer contains a large number of secretory immunoglobulin (sIgA), which is the major antibody in digestive liquids, and combines with pathogenic microorganisms and then prevents them from adhering
to mucosal epithelium [40]. Goblet cells also synthesize and secrete intestinal trefoil factor (TFF3), which is essential in maintaining mucosal integrity in gastrointestinal tract. Our results showed that MDRV caused remarkable decrease of goblet cells at the upper region of the duodenal villi, as well as cellular shrink and unclear staining with light red in the late period of MDRV infection. While HEP treatment in the infected ducklings significantly increased the goblet cells counts with normal staining at the upper region of the duodenal villi, which could efficiently maintain the secretion of intestinal mucus and mucosal integrity in intestinal tract, and then promote the repair of the injured intestinal mucosa. MDRV infection induced a significant elevated IELs and goblet cells numbers at 1 dpi–3 dpi in comparison with the blank control, which might be related to the fight against intestinal mucosal inflammation. While the two indexes significantly decreased at 6 dpi–21 dpi in MDRVinfected Muscovy ducklings, it might result from the apoptosis or necrosis of intestinal intraepithelial lymphocytes and the impairment of intestinal mucosal barrier [21,41]. However, pre-treatment of HEP in the infected ducklings could efficiently inhibit the excessive up-regulation and following reduction of IELs and goblet cells counts induced by MDRV infection from 3 dpi to 21 dpi in a timedependent manner. It was possibly associated with the reduction of
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Fig. 7. Effect of HEP on duodenal sIgA, IFN-␥ and IL-4 contents in experimental Muscovy ducklings. Duodenums were respectively collected from each group (n = 6) at 1, 3, 6, 10, 15 and 21 dpi for determination of the dynamic sIgA, IFN-␥ and IL-4 secretions using radioimmunoassay. The exam was performed in triplicate. Bars without the same superscripts (a–d) mean significant differences (P < 0.05).
inflammation at early stage of MDRV infection and the suppression of IELs and goblet cells apoptosis at late infection. Mast cells are not only the effector cells in native immunity, but also participate in acquired immunity, which are the firstline cells for anti-infection immunity. Upon contacting with the foreign antigens, mast cells can quickly and selectively produce active mediators to trigger protective immune responses [42]. And evidences have revealed that functional enhancement of mast cells can up-regulate the body’s anti-virus ability [43]. Our results showed that late infection of MDRV caused remarkable decrease of mast cells counts and cellular shrink in lamina propria of duodenal mucosa, which suggested that MDRV inhibited the growth and development of mast cells, and brought damages to intestinal native and acquired immune response. While HEP treatment in the infected ducklings significantly increased the mast cells counts with normal shape, therefore, HEP can promote the development of mast cells in small intestine to ensure the anti-virus ability of Muscovy ducklings. Collectively, HEP pretreatment not only downregulates the numbers of IELs and goblet cells in the early stage of MDRV infection to reduce the inflammation of the body, but also promote the recovery of IELs, goblet cells and mast cells in the late stage of infection to promote the repair of the intestinal mucosal immune barrier in MDRV-infected Muscovy ducklings. Mucosal immunity is the first line in the fight of body against the enteroviruses and respiroviruses. Plenty of sIgA is produced by goblet cells in the gastrointestinal tract and is the main effector in
mucosal immune response, when the viruses invade the body, they stimulate the body to secrete a large number of sIgA and then sIgA combines with the viruses adhering to mucosal surface to prevent the virus from invading epithelial cells, simultaneously plentiful mucus proteins are produced to kill the virus [44]. In our study, we also detected the effect of HEP on intestinal sIgA levels, and the results showed that the sIgA secretions in intestinal mucosa in MDRV-infected Muscovy ducklings were significantly lower than those in any of the groups, indicating that MDRV impaired the intestinal mucosal immune function in Muscovy ducklings, which is potentially associated with the destruction of goblet cells. The HEP administration in drinking water significantly increased the intestinal sIgA secretion, thus enhancing intestinal mucosal immune function and reducing MDRV-induced damages to Muscovy ducklings. Additionally, dynamic IFN-␥ and IL-4 secretions were detected in our study. IFN-␥, or type II IFN, is a representative of Th1 cytokines and plays a great role in the native and acquired immunity against viral and intracellular bacterial infections [45]. IL-4 is a kind of Th2 cytokines and can promote the proliferation and differentiation of T lymphocytes, B lymphocytes and mast cells, and also plays an important role in the formation of sIgA [46]. IL-4 has been shown to downregulate the inflammatory response through inhibition of IL-8 [47]. In the intestinal epithelia, IELs are the major source of IFN-␥, in addition, both IELs and mast cells can produce IL-4 to perform immunoregulation. Our results revealed that MDRV infection significantly suppressed the IFN-␥
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secretions at the early stage of viral infection (3–6 dpi), from 6 dpi till to 21 dpi, IFN-␥ contents in CICG gradually increased but were still lower than those in BCG. Oral administeration of HEP in the infected ducklings significantly (P < 0.05) increased the IFN-␥ secretions in comparison with the CICG, which is beneficial to recovery or enhancement of immune response in sick ducklings. Our results also showed that duodenal IL-4 contents in CICG displayed certain degrees of decrease in comparison with the BCG during the whole testing period (1–21 dpi), and pre-treatment of HEP in the infected ducklings could reverse the IL-4 decrease caused by MDRV infection and maintain the dynamic balance of IL-4 levels, which is helpful to relieve inflammation and injures of intestinal mucosal immune functions. In conclusion, our findings indicate that HEP pre-treatment can significantly improve the intestinal mucosal morphology and related parameters, the numbers of intestinal mucosal immunityrelated immunocytes as well as intestinal sIgA, IFN-␥ and IL-4 secretion in MDRV-infected Muscovy ducklings, thus repairing injures of small intestinal mucosal immune barrier, and improving immune responses and reducing the mortality rate of sick Muscovy ducklings. HEP can be used as a potential natural Chinese medicine extract for clinical prevention and control of MDRV infection and other animal diseases. In addition, we also made a preliminary exploration on the molecular mechanism of HEP in repairing the intestinal mucosa (data not shown). Our findings will provide valuable reference for clarification of the protective mechanism of HEP on intestinal mucosa and further application of HEP in control of clinical diseases. Acknowledgments This research was supported by grants from the Natural Science Foundation of China (Grant Number: 31372474) and grants from the Natural Science Foundation of Fujian Province (Grant Number: 2017J01597) References [1] V.R. Kaschula, A new virus disease of the Muscovy-duck [Cairina moschata (linn)] present in Natal, J. S. Afr. Vet. Assoc. 21 (1950) 18–26. [2] D. Gaudry, J.M. Charles, J. Tektoff, A new disease expressing itself by a viral pericarditis in Barbary ducks, C. R. Acad. Sci. Hebd. Seances Acad. Sci. D 274 (1972) 2916–2919. [3] M. Malkinson, K. Perk, Y. Weisman, Reovirus infection of young Muscovy ducks (Cairina moschata), Avian Pathol. 10 (1981) 433–440. [4] U. Heffels-Redmann, H. Muller, E.F. Kaleta, Structural and biological characteristics of reoviruses isolated from Muscovy ducks (Cairina moschata), Avian Pathol. 21 (1992) 481–491. [5] B.C. Wu, J.X. Chen, J.S. Yao, Z.H. Chen, W.L. Chen, G.P. Li, X.C. Zeng, Isolation and identification of Muscovy duck reovirus, J. Fujian Agric. Univ. 30 (2001) 227–230. [6] G. Kuntz-Simon, G. Le Gall-Reculé, C. de Boisséson, V. Jestin, Muscovy duck reovirus sigmaC protein is atypically encoded by the smallest genome segment, J. Gen. Virol. 83 (2002) 1189–1200. [7] B.C. Wu, J.X. Chen, J.S. Yao, Pathogenicity of Muscovy duck reovirus isolate B3, Chin. J. Prev. Vet. Med. 23 (2001) 422–425. [8] B.C. Wu, J.S. Yao, J.X. Chen, H.M. Lu, Z.H. Chen, Pathology of infection with reovirus isolate B3 in Muscovy ducks, J. Fujian Agric. Univ. 30 (2001) 514–517. [9] S.Y. Chen, Q.L. Hu, X.X. Cheng, S.L. Chen, B. Jiang, T.L. Lin, F.Q. Lin, X.L. Zhu, Y.Q. Cheng, Ultrastructure observation of muscovy ducks infected with Muscovy duck reovirus, Chin. J. Vet. Sci. 26 (2006) 662–664. [10] Q.X. Wang, L.J. Zhao, F. Su, B.C. Wu, G.P. Li, Effects of infection with Muscovy duck reovirus isolate B3 on the cellular immunity function of Muscovy duck, J. Fujian Agric. Univ. 38 (2009) 380–383. [11] J.X. Jiang, L.N. Huang, H.H. Jiang, J. Fang, D.B. Li, X. Cheng, Y.J. Wu, B.C. Wu, Effect of Astragalus polysaccharides on levels of cytocines in Muscovy ducks infected with Muscovy duck reovirus, Chin. Vet. Sci. 45 (2015) 873–880. [12] L.H. Zheng, Y.J. Wu, J.L. Liao, D.L. Cai, Y.Y. Zhang, E.P. Zhu, W.D. Zhou, B.C. Wu, Y.F. Huang, Chin. Vet. Sci. 2 (2017) 249–256. [13] G. Kuntzsimon, P. Blanchard, M. Cherbonnel, A. Jestin, V. Jestin, Baculovirus-expressed Muscovy duck reovirus sigmaC protein induces serum neutralizing antibodies and protection against challenge, Vaccine 20 (2002) 3113–3122.
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