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. 2024 Feb 20;103(5):103547. doi: 10.1016/j.psj.2024.103547

The effect of ghrelin on bursa and cecal tonsils of chickens infected with an attenuated virus strain of infectious bursal disease virus

Zhiyong Xu 1, Yan Yu 1,1, Shuxian Fu 1, Beibei Li 1, Liu Liu 1, Li Wang 1, Qiuxia Wang 1, Jinyou Ma 1
PMCID: PMC10912916  PMID: 38428353

Abstract

Infectious bursal disease (IBD) significantly affects the poultry industry, causing substantial economic losses. This study aimed to investigate the effects of ghrelin on chicks infected with an attenuated virus strain of IBDV (aIBDV). Chicks were divided into 3 groups: a control group (group I), an aIBDV infection group (group II), and a ghrelin + aIBDV infection group (group III). Mice in groups II and III were fed until they reached 19 d of age and then inoculated with aIBDV to establish a subclinical infection model. Group III received an intraperitoneal injection of 0.5 nmol/100 g ghrelin from d 17 to 23. The present study utilized paraffin sectioning, H&E staining, and immunohistochemical staining to examine the effects of ghrelin on the bursa of fabricius and cecum tonsils in aIBDV-infected chicks. The results indicated that at 3 d postinfection (dpi), the average body weight of group III was significantly greater than that of group II (P < 0.05). At 3 and 7 dpi, the proportion of large lymphoid follicles in the bursa of fabricius in group III was notably greater than that in group II (P < 0.05). aIBDV infection resulted in bleeding, edema, and fibrosis in the cecal mucosal layer of chicks, but ghrelin administration mitigated these pathological changes. At 3 and 7 dpi, the thickness of the lamina propria in the cecal tonsils of group III was significantly lower than that in the cecal tonsils of group II (P < 0.05). Additionally, the percentage of large lymphoid follicles in the cecal tonsils of group III was significantly greater than that in group II at 3 and 5 dpi (P < 0.05). There were significantly fewer macrophages in the cecal tonsils of group III than in those of group II at 1, 3, and 5 dpi (P < 0.05). In conclusion, ghrelin supplementation improved performance and mitigated bursal atrophy in aIBDV-infected chicks. It also reduced histological lesions and immune responses in the cecum tonsil. Notably, the reduction in macrophages in the cecum tonsil following ghrelin administration may decrease the risk of aIBDV spread.

Keywords: ghrelin, attenuated IBDV, cecal tonsil, macrophage, chick, bursa of fabricius

INTRODUCTION

Infectious bursal disease virus (IBDV) is a nonenveloped double-stranded RNA virus that primarily affects the bursa of fabricius (BF). Infection with IBDV in chicks can lead to necrosis and apoptosis of B lymphocytes in the BF, resulting in severe and long-term immunosuppression (Sharma et al., 2000; He et al., 2012; Huang et al., 2021). There are 2 serotypes of IBDV, with serotype I being pathogenic and serotype II not causing disease in chickens. The pathogenic serotype I field isolates are classified into classical virulent (cv), very virulent (vv), and variant strains (Müller et al., 2003). While vv IBDV infections have been gradually brought under control through improved feeding management and widespread vaccine usage, subclinical infections caused by variant IBDVs have become prevalent in major chick-farming regions of China (Fan et al., 2019). These strains do not cause significant clinical morbidity or mortality in chicks, which often leads to them being overlooked. However, they can still cause bursa atrophy, maternal antibody disruption, and growth retardation, making IBD more insidious and harmful (Wang et al., 2022). Therefore, identifying methods to reduce the occurrence of subclinical IBDV infections is highly important.

Infectious bursal disease is a contact infectious disease primarily transmitted orally through direct or indirect contact with IBDV-contaminated feed, drinking water, utensils, and other sources. Following oral infection, IBDV enters the bloodstream and initially replicates in gut-associated macrophages and lymphoid cells before it reaches the BF. The virus then replicates in proliferating B lymphocytes of the BF, leading to pathological changes in the organ (Müller et al., 1979). Avian cecal tonsils (CT) are the largest lymphoid aggregates of avian gut-associated lymphoid tissue (GALT) and play a significant role in the immune response (Heidari et al., 2014). Müller et al. (1979) demonstrated that IBDV viral antigen can be detected as early as 4 h after oral infection and is mainly associated with macrophages and, to a lesser extent, with lymphoid cells of the CT and other regions of the cecum. These findings suggested that macrophages play a vital role in the spread of IBDV. Therefore, investigating the immune response and changes in macrophage populations in the CT can contribute to a better understanding of IBD occurrence in chickens following IBDV infection.

Chicken ghrelin, a peptide hormone consisting of 26 amino acids, is secreted by enteroendocrine cells in the proventriculus of chickens (Kaiya et al., 2002). It is widely expressed in various organs, including the brain, lung, heart, intestine, immune cells, and pancreas (Ma et al., 2015; Mehdar KM., 2021). Studies have shown that chicken ghrelin functions similarly to ghrelin in mammals, stimulating GH release and promoting gastrointestinal contraction. However, there are differences in lipid metabolism, energy sources, and food intake (Kaiya et al., 2009; Kaiya et al., 2013). In the immune system, researchers have shown that ghrelin in mammals can promote the proliferation of T and B lymphocytes (Koo et al., 2001), regulate the expression of cytokines (Li et al., 2004), exert anti-inflammatory effects (Matuszyk et al., 2016), and protect organs from damage (Sukumaran et al., 2018). Previous research has shown that the plasma ghrelin concentration increases after IBDV infection and then decreases. Additionally, the mRNA expression levels of ghrelin and ghrelin-related genes, ghrelin-o-acyltransferase, and growth hormone secretagogue receptor in the bursa also increase during IBDV infection but subsequently decrease (Yu et al., 2019). Chicken ghrelin may alleviate bursal injury by reducing cytokine production in the bursa after vvIBDV infection (Yu et al., 2020), suggesting its potential to function similarly to mammalian ghrelin in the immune system and inhibit acute inflammation caused by viruses such as vvIBDV. However, the effect of ghrelin on IBDV subclinically infected chickens has not been reported. This study aimed to investigate the effect of ghrelin on the BF and CT in chickens with subclinical IBDV infection using an attenuated virus strain of IBDV (aIBDV). The results of this study may contribute to the clinical application of ghrelin.

MATERIALS AND METHODS

Cultivation and Amplification of aIBDV

The IBDV BC6/85 strain (1 μg, China Veterinary Drug Control Institute, Beijing, China) was diluted with sterile PBS (pH = 7.38) at a ratio of 1:100. The diluted strain was subsequently inoculated into the chorioallantoic membrane of specific pathogen-free (SPF) chicken embryos (supplied by Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) after 9-11 d of incubation at a dose of 0.1 mL/embryo. After 72 h, the live chicken embryos were euthanized by cooling at 4°C for 4 h. The chorioallantoic membrane was collected aseptically and ground with liquid nitrogen. The supernatant was obtained by centrifuging the ground membrane after 3 cycles of freezing and thawing. A portion of the supernatant was subjected to virus RNA extraction, while the remaining portion was utilized for subsequent experiments. SPF chickens aged 19 to 21 d were infected with the aforementioned supernatant through anal wiping at a dose of 0.1 mL/embryo. Three days after infection, the BFs were promptly harvested and ground into powder. The supernatant was then collected after centrifugation. Like in the previous step, one portion of the supernatant was used for virus RNA extraction, while the other portion was utilized for subsequent experiments.

Animal Culture and Sample Collection

One hundred and twenty 1-day-old Jinghong laying chickens, provided by HULAN BIO COMPANY in Xinxiang, Henan, were housed in negative-pressure isolators (GJ-1; Suzhou Fengshi Laboratory Animal Equipment Co., Ltd.) at a specific pathogen-free animal laboratory. The temperature and pressure conditions were as follows: 1 to 3 d, 35 to 36°C, 25 Pa; 4 to 7 d, 32 to 35°C, 25 to 35 Pa; 8 to 14 d, 29 to 32°C, 35 to 45 Pa; 15 to 21 d, 21 to 25°C, 55 to 75 Pa; and 22 to 27 d, 21 to 25°C, 55 to 75 Pa. The humidity levels were 65 to 70% for d 1 to 10 and 60 to 65% for d 11 to 27. The chickens were fed fodder purchased from Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China) and had access to sterile water ad libitum.

On the 17th d of rearing (D17), the chickens were weighed and randomly divided into 3 groups (n = 20+20). From d 17 (D17) to D23, one group of chickens (group III) was intraperitoneally injected with 0.5 nmol of ghrelin/100 g body weight (BW) (Abcam, ab120231), as per a previous study by Yu et al. (2021). On D19, the second group of chickens (group II) and group III were inoculated with the above supernatant through an anal wipe at a dose of 0.1 mL/chicken. The third group (group I) was mock-treated with sterile PBS (pH = 7.38). The chickens in groups II and III were housed in different negative-pressure isolators in one room, while the chickens in group I were housed in different negative-pressure isolators in another room. The specific group assignments are shown in Table 1.

Table 1.

Grouping and treatment of experimental chickens.

Groups Control group (no infection, no treatment) Infected group (The chickens were infected with aIBDV, no treatment) Treatment group (The chickens were infected with aIBDV, and treated with ghrelin)
Number of chickens 40 40 40
Group symbols I II III
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From 1 dpi to 7 dpi, ten chickens were randomly selected from each group (5 chickens per pen), weighed, and then euthanized via cervical dislocation under deep nembutal anesthesia (45 mg/g of BW, intraperitoneal injection; Shanghai Chemical Factory, Shanghai, China). The BF and CT were promptly collected from each chicken and fixed in 4% paraformaldehyde (Solarbio, P1110, Beijing, China) for hematoxylin and eosin (H&E) staining and immunohistology staining. All animal experiments in the study were approved by the scientific ethical committee of the Henan Institute of Science and Technology (Permit Number: 2020HIST016). This experiment was repeated 3 times.

Viral RNA Extraction and Agarose Gel Electrophoresis

Viral RNA extraction was performed using a viral RNA extraction kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The extracted RNA was then reverse transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent kit (TaKaRa). PCR amplification was carried out using 2X Ex Taq Master Mix (Takara Bio). The amplified products were analyzed via 1.5% agarose gel electrophoresis.

Hematoxylin and Eosin Staining

BF and CT tissues were fixed in 4% paraformaldehyde and prepared as paraffin sections with a thickness of 5 μm. The sections were dewaxed in xylene, rehydrated in gradient alcohol, and stained in hematoxylin solution for 5 min. Subsequently, the sections were dehydrated in gradient alcohol with eosin dye in 85% ethanol, followed by transparency in xylene. The sections were then sealed with neutral gum, observed, and photographed under a microscope. The size of the lymphoid follicles in the BF, the number of macrophages in the CT, the thickness of the lamina propria, and the area of lymphoid follicles in the CT were measured using Image-Pro Plus 6.0 software.

Immunohistochemistry

The deparaffinized sections of the BF were subjected to antigen retrieval in 10 mM citrate buffer at 95 to 98°C for 20 min. Subsequently, quenching of endogenous peroxidase activity was performed by incubating the sections in a 3% H2O2/methanol bath for 15 min. After several washes with PBS (pH 7.4), the sections were incubated with 5% normal goat serum at room temperature for 20 min and then with a mouse anti-VP3 primary antibody (1:200; Biorbyt, ARB23535) at 4°C overnight. The sections were rinsed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (1:200; Absin, ABS20039) for 2 h. Immunoreactivity was visualized using a DAB chromogenic kit (ZSGB-Bio, ZLI-9018, Beijing, China). The number of VP3-positive cells was measured in each section using Image-Pro Plus 6.0 software.

Body Weight Statistics

At each time point, the live weight (g) of 6 chicks in each group was measured and averaged, after which the standard deviation was calculated.

Bursa Index Statistics

At each time point, the bursa weights (mg) of 6 chicks from each group were measured and recorded. The bursa index of each chick was calculated using the formula: Bursa index (mg/g) = bursa weight (mg)/chick live weight (g). The average value of the bursa index was determined, and the standard deviation was calculated.

Number Statistics of Large Lymphoid Follicles in the Fabricius and Cecum

At each time point, H&EH&E staining was performed on 6 discontinuous sections of the bursa and cecal tonsils from 6 chicks in each group. Image-Pro Plus 6.0 software was used to calculate the follicular area (μm2) in the bursa and cecal tonsils under a 400x lens. In this study, large follicles were defined as bursal lymphatic follicles with an area of more than 60,000 square microns and tonsillar cecal follicles with an area of more than 30,000 square microns. The percentage (%) of large follicles relative to the total number of follicles in each group was calculated.

Thickness Statistics of Cecal Tonsil Lamina Propria

At each time point, H&E staining was performed on 6 discontinuous sections of the cecal tonsils from 6 chicks in each group. Image-Pro Plus 6.0 software was used to calculate the thickness of the lamina propria of the cecal tonsil under a 400x lens, and its standard deviation was calculated.

Cecal Tonsil Macrophage Number Statistics

At each time point, H&E staining was performed on six discontinuous sections of the cecal tonsils from six chickens in each group. The number of cecal tonsil macrophages in a single field of vision under a 400x lens was calculated using Image-Pro Plus 6.0 software. The average value of the calculated results was taken, and the standard deviation was calculated.

Statistical Analysis

The area of lymphoid follicles in the BF and CT, the thickness of the lamina propria, and the number of macrophages in the CT were measured using Image-Pro Plus 6.0 software. SPSS software (IBM SPSS Statistics 22) was used to analyze the differences among multiple groups using one-way analysis of variance (ANOVA). The measurement data are presented as the mean ± SD. A p value of < 0.05 was considered to indicate statistical significance.

RESULTS

Cultivation and Amplification of aIBDV

After the BC6/85 strain was passed through chicken embryos and SPF chickens, viral RNA was extracted from the chorioallantoic membrane, and the viral gene was amplified via PCR. A clear band of 216 bp was observed in the 1.5% agarose gels, which corresponded to IBDV VP2 mRNA according to the primer used (Figure 1A). SPF chickens were inoculated with the supernatant of the chorioallantoic membrane on the 14th d of rearing. No obvious clinical symptoms, such as depression, ruffled feathers, huddling, respiratory distress, or diarrhea, were observed. However, the size of the chicken BFs was smaller than that of the control, and a clear 216 bp band was present in the bursal tissue according to the PCR assay (Figure 1B). These results indicated that the virulence of the BC6/85 virus was weakened when inoculated into chicken embryos and that the attenuated virus could infect chickens without causing obvious clinical symptoms.

Figure 1.

Figure 1

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Expression of VP2 in chick embryo chorioallantoic membrane and SPF chick bursal. (A) Chicken embryo chorioallantoic membrane VP2 expression was analyzed by PCR. (B) PCR analysis of VP2 expression in the BF of SPF chickens. The amplification of β-actin was used as an internal control. (M, Marker. 1, β-actin).

Ghrelin Inhibited the Expression of VP3-Positive Cells in Mice Infected With aIBDV

VP3 is a structural protein that forms the IBDV capsid, and its expression level is often used to represent the viral load (Yu et al., 2020). Following infection with aIBDV, all chickens in the different treatment groups showed no obvious clinical signs. However, VP3-positive cells were observed in the BF of chickens in group II and group III at 1, 3, 5, and 7 dpi using immunohistochemical staining (Figure 2A), while no VP3-positive cells were observed in group I (Figure 2A [a]), indicating that the chickens were infected with aIBDV. At 1, 3, 5, and 7 dpi, the mean integrated optical density (IOD) of VP3-positive cells in the bursa of fabricius in group II and group III initially increased and then gradually decreased. The mean integrated optical density was highest at 3 dpi. Additionally, at 1, 3, 5, and 7 dpi, the mean integrated optical density of VP3-positive cells in the bursa of fabricius in group III was significantly lower than that in group II (P < 0.05 or P < 0.01) (Figure 2B).

Figure 2.

Figure 2

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VP3 expression in the bursal of aIBDV-infected chickens. (A) VP3-positive cells were detected by immunohistochemical staining (a, I group. b, 1 dpi-II group. c. 1 dpi-III group. d, 3 dpi-II group. e, 3 dpi-III group. f, 5 dpi-II group. g, 5 dpi-III group. h, 7 dpi-II group. i, 7 dpi-III group. 400✕). (B) Mean integrated option density of VP3-positive cells. Compared to the group II, *P<0.05, **P<0.01.

Ghrelin Prevented the Reduction in Chicken Weight Gain Caused by aIBDV Infection

Weight gain is a crucial indicator for assessing the health of animals. In this study, the weight gain of chickens was analyzed across different treatment groups. At 1 dpi, the average weight of the chickens in group I was 163.55 ± 4.09 g, while it was slightly lower in group II (156.26 ± 6.04 g) than in group I, although the difference was not statistically significant (P > 0.05). The weight of group III was significantly greater than that of group II, measuring 175.61 ± 7.55 g (P < 0.05). At 3 dpi, the weight of the chickens in group II was lower than that of the chickens in groups I and III, measuring 155.35 ± 7.55 g (P < 0.05). At 5 dpi, the chickens in group II exhibited rapid growth, with an average weight of 231.15 ± 11.72 g. Although the weights of the chicks in groups I and III were greater than those in group II, there were no significant differences among the 3 treatment groups (P > 0.05) at 5 and 7 dpi (Fig. 3).

Figure 3.

Figure 3

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Changes in body weight of aIBDV-infected chickens treated with ghrelin administration. Compared to the group I, #P<0.05. Compared to the group II, *P<0.05.

Ghrelin Alleviated the Descending Tendency of the Bursal Index Induced by aIBDV

The bursal indices of chickens in the different groups were analyzed, revealing that the bursal indices in group II were lower than those in group I and group III at 1 dpi, and a significant difference was observed between groups II and III (P < 0.05). The bursal indices were 4.29 ± 0.89 mg/g, 3.61 ± 0.36 mg/g, and 4.80 ± 0.79 mg/g in group I, group II, and group III, respectively. At 3 dpi, the bursal indices in group II were lower than those in group I and group III, but these 2 groups were not significantly different (P > 0.05). Furthermore, there was no significant difference in the bursal indices among the 3 treatment groups at 5 and 7 dpi (P > 0.05). In summary, chickens infected with aIBDV exhibited a decrease in the bursal index at 1 dpi and 3 dpi, while administration of ghrelin alleviated this decreasing trend during the early infection stage (Figure 4).

Figure 4.

Figure 4

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Effect of ghrelin on bursal index of chickens infected with aIBDV. Compared to the group II, *P<0.05.

Ghrelin Enhanced the Percentage of Larger Lymphoid Follicles in the BF of Chickens Infected With aIBDV

Six nonadjacent sections of each chicken BF were stained with H.E. (Figure 5A), and the lymphoid follicle area of the BF was measured and analyzed. The results indicated that at 1 dpi, 3 dpi, 5 dpi, and 7 dpi, the percentage of large bursal lymphoid follicles in group II (36.96 ± 3.20, 39.17 ± 3.28, 41.26 ± 2.98, 45.48 ± 3.02) was significantly lower than that in group I (54.88 ± 3.56, 55.81 ± 4.12, 58.14 ± 2.90, 55.76 ± 5.30) (P < 0.05). Compared to those in group II, the percentages of large bursal lymphoid follicles in group III were lower (40.55 ± 2.60, 47.74 ± 2.11, 49.00 ± 2.20, and 48.78 ± 2.80) and reached significant differences at 3 dpi and 7 dpi (P < 0.05) (Figure 5B).

Figure 5.

Figure 5

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Effect of ghrelin on BF lymphoid follicle of aIBDV-infected chickens. (A) Lymphoid follicle HE staining of the bursal of the chickens (a, 1 dpi-I group. d, 3 dpi-I group. g, 5 dpi-I group. j, 7 dpi-I group. b, 1 dpi-II group. e, 3 dpi-II group. h, 5 dpi-II group. k, 7 dpi-II group. c, 1 dpi-III group. f, 3 dpi-III group. i, 5 dpi-III group. l, 7 dpi-III group. 400✕), “←” indicates a large bursal lymphoid follicle. (B) Dynamic changes of the large lymphoid follicle in chicken BF. Compared to the group I, #P<0.05. Compared to the group II, *P<0.05.

Histopathological Changes in the Cecal Tonsil

In comparison to group I, group II exhibited evident changes at 1 d postinfection, characterized by edema and widening of the tissue space in the cecal mucosal layer, along with bleeding in the lamina propria. Similar bleeding in the lamina propria was observed in group III. By day 3 postinfection, group II displayed further exacerbation of cecal mucosal edema and widening, accompanied by hemorrhage in the lamina propria, infiltration of inflammatory cells and a reduction in the number of cells within the diffuse lymphoid tissue. Group III exhibited analogous symptoms but to a lesser extent. On the fifth day postinfection, group II still presented a limited amount of bleeding in the cecum lamina propria, intertissue fibrosis and inflammatory cell infiltration. In contrast, group III demonstrated reduced fibrosis and a decreased presence of inflammatory cells. By the seventh day postinfection, significant fibrosis and inflammatory cell infiltration were evident in the lamina propria of the cecum in group II, while group III displayed a milder degree of fibrosis and inflammatory cell infiltration (Figure 6).

Figure 6.

Figure 6

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Effect of ghrelin on the histological structure of cecal tonsils of aIBDV-infected chickens (a, 1 dpi-I group. d, 3 dpi-I group. g, 5 dpi-I group. j, 7 dpi-I group. b, 1 dpi-II group. e, 3 dpi-II group. h, 5 dpi-II group. k, 7 dpi-II group. c, 1 dpi-III group. f, 3 dpi-III group. i, 5 dpi-III group. l, 7 dpi-III group. 400✕). “ ←” indicates hemorrhage, “ ” indicates edema, “ ” indicates fibrosis, and “ ” indicates inflammatory cells.

Ghrelin Reduced the Thickness of the Lamina Propria of the CT in Chickens Infected With aIBDV

Discontinuous sections of the cecal tonsils from the chickens in each group were subjected to H&E staining (Figure 7A), and the lamina propria thickness was quantified, considering the maximum value in each group for statistical analysis. The results revealed a significant increase in CT lamina propria thickness in group II (1,543.29 ± 43.84, 1,783.52 ± 97.11) at 3 and 7 d postinfection (dpi) compared to that in group I (1,343.47 ± 71.32, 1,125.89 ± 232.44) (P < 0.05). Conversely, compared with those in group II, the CT lamina propria thickness in group III (1400.55 ± 28.52, 1535.41 ± 63.72) was significantly lower at 3 and 7 dpi (P < 0.05) (Fig. 7B).

Figure 7.

Figure 7

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Changes in the thickness of lamina propria in CT of aIBDV-infected chickens treated with ghrelin administration. (A) HE staining of the chicken CT (a, 1 dpi-I group. d, 3 dpi-I group. g, 5 dpi-I group. j, 7 dpi-I group. b, 1 dpi-II group. e, 3 dpi-II group. h, 5 dpi-II group. k, 7 dpi-II group. c, 1 dpi-III group. f, 3 dpi-III group. i, 5 dpi-III group. l, 7 dpi-III group. 400✕), “ ” indicates the thickness of lamina propria of the CT. (B) Dynamic changes in the thickness of lamina propria of chicken CT. Compared to the group I, #P<0.05. Compared to the group II, *P<0.05.

Ghrelin Increased the Percentage of Larger Lymphoid Follicles on CT Images of Chickens Infected With aIBDV

Five nonconsecutive sections of the cecal tissue from each group were stained with H&E (Figure 8A), and the area of the large lymphoid follicles in each section was calculated using IPP software. At 1, 3, 5, and 7 dpi, the percentage of large lymphoid follicles in the cecal tissue of group II (40.91 ± 3.00, 39.33 ± 2.09, 29.09 ± 1.60, 38.63 ± 2.90) was significantly lower than that in group I (52.63 ± 3.32, 60.38 ± 3.30, 41.08 ± 2.20, 50.00 ± 4.10) (P < 0.05). Furthermore, compared to that in group II, the percentage of large lymphoid follicles in group III (48.44 ± 2.80, 32.78 ± 1.26) increased and reached a significant difference at 3 and 5 dpi (P < 0.05).

Figure 8.

Figure 8

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Changes in CT lymphoid follicle in aIBDV-infected chickens after ghrelin administration. (A) HE staining of lymphoid follicle in the chicken CT (a, 1 dpi-I group. d, 3 dpi-I group. g, 5 dpi-I group. j, 7 dpi-I group. b, 1 dpi-II group. e, 3 dpi-II group. h, 5 dpi-II group. k, 7 dpi-II group. c, 1 dpi-III group. f, 3 dpi-III group. i, 5 dpi-III group. l, 7 dpi-III group. 400✕), “ ←” indicates a large bursal lymphoid follicle. (B) Dynamic changes of the lymphoid follicle in chicken CT. Compared to the group I, #P<0.05. Compared to the group II, *P<0.05.

Ghrelin Decreased the Number of Macrophages in CT Induced by aIBDV

The macrophage count in the CT was determined using IPP (Figure 9). Compared to those in group I (11.16 ± 2.12, 10.70 ± 1.22, 10.55 ± 2.44, 9.93 ± 1.33), the number of macrophages in the cecal amygdala in group II (21.87 ± 3.20, 18.45 ± 2.16, 17.75 ± 1.66, 16.60 ± 2.98) significantly increased at 1, 3, 5, and 7 dpi (P < 0.05). Moreover, compared with those in group II, the number of macrophages in the cecal amygdala in group III (15.20 ± 1.17, 14.20 ± 1.20, 12.40 ± 1.52) significantly decreased at 1, 3, and 5 dpi (P < 0.05).

Figure 9.

Figure 9

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Changes in CT macrophages in aIBDV-infection chickens after ghrelin administration. (A) HE staining of the chicken CT (a, 1 dpi-I group. d, 3 dpi-I group. g, 5 dpi-I group. j, 7 dpi-I group. b, 1 dpi-II group. e, 3 dpi-II group. h, 5 dpi-II group. k, 7 dpi-II group. c, 1 dpi-III group. f, 3 dpi-III group. i, 5 dpi-III group. l, 7 dpi-III group. 400×), “ ←” indicates the macrophages. (B) Dynamic changes in the number of macrophages of chicken CT. Compared to the group I, #P<0.05. Compared to the group II, *P<0.05.

DISCUSSION

Infectious bursal disease is a highly significant immunosuppressive disorder that poses a serious threat to poultry farming worldwide. Since 2017, subclinical infections caused by variant strains of IBDV caused by several chicken farms in eastern China have been reported (Fan et al., 2019). These subclinical infections often go undetected because they do not result in clinical morbidity or mortality. However, they can lead to significant bursal atrophy and subsequent immunosuppression, which in turn increases the susceptibility of chickens to secondary infections by other pathogens and causes significant economic losses (Aliyu et al., 2022; Wang et al., 2022). Therefore, it is crucial to study ways to reduce the occurrence of subclinical IBDV infections. In this study, Jinghong laying hens infected with aIBDV did not exhibit any clinical symptoms, such as depression, ruffled feathers, huddling, respiratory distress, or diarrhea. However, they showed slow weight gain, a decreased bursal index, and the presence of VP3 immunopositive cells in the bursa of fabricius. These results confirm the successful establishment of a subclinical infection model.

Body weight can serve as an indicator of chicken performance to some extent. Therefore, our investigation focused on the effect of ghrelin on the body weight of chickens infected with aIBDV. Previous studies have demonstrated the significant upregulation of the mRNA expression of cytokines, including interleukin (IL)-6, IL-1β, and IL-8, after chickens are infected with IBDV. However, in the group treated with ghrelin, there was a significant decrease in the expression of the IL-6, IL-1β, and IL-8 mRNAs. Furthermore, ghrelin administration resulted in a significant reduction in bursal injury and prevented the decrease in bird weight gain caused by IBDV (Yu et al., 2020). In this experiment, ghrelin significantly mitigated the negative effect of aIBDV infection on weight gain in chickens at 1 dpi and 3 dpi, as shown in Figure 3. One possible explanation is that when chickens are infected with aIBDV, their bodies mount an inflammatory response to combat pathogen invasion (Liu et al., 2010), which in turn hampers chicken growth and slows weight gain. Additionally, ghrelin administration reduced the decrease in chicken weight gain caused by aIBDV infection, possibly by suppressing the inflammatory response upon ghrelin supplementation (Yu et al., 2020), leading to an increase in appetite. These findings are consistent with previous results showing the effect of ghrelin on the body weight of IBDV-infected chickens (Yu et al., 2020).

The BF is not only a primary immune organ unique to chickens but also a target of IBDV attack (Ingrao et al., 2013). Therefore, changes in the BF can be used as indicators of the effects of aIBDV on chickens. In this study, the percentage of larger lymphoid follicles in the BF decreased after chicken infection with aIBDV, suggesting the occurrence of damage, such as bursal atrophy and dehydration, in aIBDV-infected chickens. This damage is considered a pathological change resulting from IBDV infection (Aihara et al., 2015; Huang et al., 2021). Moreover, a study demonstrated that ghrelin mitigated bursal fibrosis in SPF chicks infected with IBDV by reducing the number of inflammatory cells and downregulating the expression of TGF-β and MMP-9 (Yu et al., 2021). In the present study, ghrelin administration significantly increased the bursal index at 1 dpi (P < 0.05), enhanced the percentage of larger lymphoid follicles, and reduced the percentage of smaller lymphoid follicles in the BF compared to those in the II group. These results indicate that ghrelin administration mitigated BF damage and protected the original lymphoid follicles in chickens infected with aIBDV. These findings are consistent with previous reports (Işeri et al., 2005; Fahim et al., 2011; Çimen et al., 2019). The mucosal layer of the cecum consists of the mucosal epithelium, lamina propria, and mucosal muscle layer. Within this layer, the cecal tonsil is primarily found in the lamina propria. It is composed predominantly of diffuse lymphoid tissue and lymphatic follicles, making it the largest lymphocyte aggregate in avian GALT. The cecal tonsil plays a crucial role in providing immune responses to bacterial, viral, and parasitic infections (Heidari et al., 2014).

IBDV, a virus that replicates in B lymphocytes, initially invades B lymphocytes within follicles. This invasion leads to an increase in B lymphocyte necrosis and a reduction in the follicle area. Zhao et al. (2016) conducted a study that revealed how IBDV infection can cause necrosis, disappearance, and vacuolation of numerous lymphocytes within the tonsillar lamina propria of the cecum, resulting in a decrease in lamina propria thickness. However, the effect of weakened IBDV on the follicles and surrounding tissue structure in the cecal amygdala has not been studied.

In this study, histological observation demonstrated significant congestion, bleeding, interstitial edema, and an abundance of inflammatory cells in the cecal lamina propria tissue of chicks infected with aIBDV compared to those in the control group (I). The number and distribution of diffuse lymphocytes were reduced, and the lesions were sparser. Vacuoles were present in the nodules of the lymph nodes, and B-lymphocytopenia was observed. Treatment with ghrelin resulted in a reduction in the degree of cecal lamina propria edema, a decrease in the number of inflammatory cells, and a decrease in the number of vacuoles. It also led to an increase in the number of lymphocytes in the diffuse lymphoid tissue.

Furthermore, the results showed that the cecal lamina propria thickness was greater in the aIBDV group than in the control group (I). However, after ghrelin treatment, the cecal lamina propria thickness in the ghrelin treatment group was significantly lower than that in the aIBDV group. This contradicts the findings of Zhao et al. (2016), suggesting that the different effects of strong and weak IBDV on the swelling of the cecal lamina propria may be the reason for the discrepancy. Moreover, although the cecal lamina propria thickened after IBDV infection, the number of B lymphocytes in lymphoid nodules and plasma cells in diffuse lymphoid tissue decreased. This decrease resulted in a significant reduction in the proportion of large lymphoid follicles. These observations indicate that aIBDV infection inhibits the immune ability of the cecal tonsil, consistent with the effects of IBDV on the cecal tonsil.

Various studies have demonstrated the strong anti-inflammatory and healing effects of ghrelin. Matuszyk et al. (2016) reported that ghrelin exerts these effects on acetic acid-induced colitis in rats. Ghrelin has also been shown to reduce tissue damage in rats after experimental partial unilateral ureteral obstruction due to its anti-inflammatory and antioxidant properties (Çimen et al., 2019). Additionally, ghrelin enhances the regenerative potential of gastrointestinal epithelia in doxorubicin-damaged mice and may alleviate gastric oxidative damage through its antioxidant and anti-inflammatory properties (Işeri et al., 2005; Fahim et al., 2011).

In the present study, the administration of ghrelin significantly reduced the thickness of the lamina propria and increased the proportion of large lymphoid follicles. This is likely due to the addition of ghrelin weakening the edema and inflammation induced by aIBDV in the cecal tonsil, thereby reducing its thickness (Işeri et al., 2005; Fahim et al., 2011; ). Moreover, ghrelin mitigated the damage to B lymphocytes in lymphoid follicles and increased the percentage of larger lymphoid follicles.

Macrophages serve as the primary cells responsible for the innate immune response in host organisms. While B lymphocytes are typically targeted by IBDV, it has been observed that IBDV can also replicate and proliferate within macrophages. R. Muller conducted a study in which chickens infected with IBDV through the digestive tract first replicated in gut-associated macrophages and subsequently entered the bloodstream to the BF, resulting in pathological changes in the BF (Müller et al., 1979). Our own findings indicated that the number of macrophages in the CT of IBDV-infected chickens increased, but administration of ghrelin was able to reduce this number in aIBDV-infected chickens. Consequently, while ghrelin administration reduced the immune response of the host, it also decreased the risk of aIBDV spreading in the early stages of infection, which aligned with our results showing that the number of aIBDV- and VP3-immunopositive cells was lower in the BF of group III chickens than in that of group II chickens.

In this study, we successfully generated a subclinical infection model by attenuating the virulence of the IBDV BC6/85 strain through passaging in chicken embryos. We investigated the effects of attenuated IBDV, which does not cause clinical morbidity or mortality in chickens, on immune organs such as the BF and CT. We observed that exogenous ghrelin reduced the negative effects of aIBDV infection in chickens, as indicated by the slow weight gain, reduction in bursal index, and increase in the proportion of large lymphoid follicles. These findings suggest that ghrelin administration can improve the production performance of aIBDV subclinically infected chickens while minimizing damage to the BF. In the CT, aIBDV infection led to an increase in lamina propria thickness and a decrease in the proportion of large lymphoid follicles, while exogenous ghrelin reduced the immune response in this area, consistent with its known role in the immune system of mammals. This study represents the first investigation into the effect of ghrelin on the BF and CT in chickens infected with aIBDV, providing a basis for the potential application of ghrelin in treating subclinical infection caused by aIBDV.

ACKNOWLEDGMENTS

This research was supported by National Natural Science Foundation of China-Henan Joint Fund (Grant No. U1904117), International Science and Technology Cooperation Project in Henan (Grant No. 232102520010), and the Henan Provincial Science and Technology Research Project (No. 222102110348, 232102310480).

Authors Contribution: Zhi-yong Xu carried out most of the experiments, wrote the manuscript, and should be considered as the first author. Yan Yu critically revised the manuscript and the experiment design. Shu-xian Fu, Bei-bei Li, Liu Liu, Li Wang, Qiu-xia Wang and Jin-you Ma helped with the experiment. All the authors read and approved the final version of the manuscript.

DISCLOSURES

We declare that we have no conflict of interest.

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