Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 57 issue 6 (june 2023) : 736-741

Cytokine Gene Expression and IgA Production in the Spleen of Chickens Infected with Eimeria tenella

Liushu Jia1, Bianhua Zhou1,*, Hongwei Wang1, Fan Yang1, Guoyong Wang1, Tao Kong1
1College of Animal Science and Technology, Henan University of Science and Technology, Kaiyuan Avenue 263, Luoyang, Henan 471000, People’s Republic of China.
Cite article:- Jia Liushu, Zhou Bianhua, Wang Hongwei, Yang Fan, Wang Guoyong, Kong Tao (2023). Cytokine Gene Expression and IgA Production in the Spleen of Chickens Infected with Eimeria tenella . Indian Journal of Animal Research. 57(6): 736-741. doi: 10.18805/ijar.B-1188.
To explore the effect of Eimeria tenella infection on the cytokines gene expression and IgA production in the spleen of chickens, the morphological characteristics of the spleen were observed through optical and transmission electron microscopy. The IgA production was determined through immunohistochemistry. The mRNA expression levels of splenic cytokines were detected through real-time PCR. Compared to the control group, along with the infection of E. tenella, the splenic lymphocytes exhibited irregular and cracked membranes, mitochondria swelled even vacuolization, the IgA expression in spleen tissue was decreased by 55.57% (p<0.01). Likewise, the mRNA expression levels of IL-2 and IL-1β decreased by 40% (p<0.01) and 43% (p<0.05), respectively. By contrast, the IL-6, IFN-γ and IL-10 levels increased by 158% (p<0.01), 464% (p<0.05) and 379% (p<0.01), respectively. These results indicated that the spleen implement an important function in the antagonism of E. tenella, which suggest a new strategy to control coccidiosis by improving the peripheral immunity of chickens. 
Coccidiosis of domestic fowl is caused by Eimeria spp. which mainly invade the intestinal mucosal epithelium of chickens (Lobago et al., 2005; Shirley and Lillehoj, 2012). Eimeria tenella, as one of the most virulent strains of genus Eimeria, inhabit in the caecum of chicken, leading to severe disease or death. Meanwhile, Eimeria spp. infecting chickens stimulate the avian immune system and control intestinal parasitism (Yun et al., 2000).

The spleen is considered as the most important immune organ that helps the body filter the blood and fight infection because of its specialised structure in combination with a highly organised lymphoid compartment (Springer, 1995; Serbina et al., 2003; Mebius and Kraal, 2005; Bronte and Pittet, 2013; Ren et al., 2019). Adaptive immunity, as well as innate immunity (Min et al., 2013; Tian et al., 2014), protects hosts against coccidial infections (Yun et al., 2000; Boulton et al., 2018). For example, infected chickens produce three classes of antibodies, namely, IgA, IgY and IgM (Sun et al., 2012), in response to Eimeria spp. challenge. Amongst these antibodies, IgA is the most important (Geuking et al., 2012; Brandtzaeg, 2013); IgA can be divided into two types according to their immune function, namely, serum free type and secretory type. The spleen is an important site where IgA B-cells proliferate and differentiate because of the structure and location of this organ in systemic circulation; this organ is also complicated and regulated by cytokines and chemokines. Cytokines, such as interleukin-1 (IL-1) (Ben-Sasson et al., 2009), IL-2 (Bell et al., 2015; Yang et al., 2016), IL-6 (Ramsay et al., 1994; Zhong et al., 2019), IL-10, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) (Reis et al., 2011; Dorhoi and Kaufmann, 2014), are factors necessary to develop IgA-producing B-cells. These B-cells either remove invading microorganisms by developing neutralising antibodies or eliminate viruses by activating cytotoxic T-cells (Joosten et al., 2013). Singh et al., (1999) and Bansal et al., (2010) isolated splenic cells, Peyer’s patches or lamina propria cells from mice previously immunised with Salmonella typhi porins and observed that the cytokine levels of IL-1, IL-2, IFN-γ and IL-4 in the isolated cells significantly increase. The expression levels of IFN-γ, IL-10 and TGF-β4 also increase in the spleen of chickens infected with Ascaridia galli (Pleidrup et al., 2014). However, the spleen-related immune function of E. tenella-infected chickens has been rarely reported.

This study investigated the morphological characteristics and IgA production in the spleen tissue of chickens. The related cytokine gene expression in the spleen was also determined. This study also described the peripheral immunity of the spleen reacting to E. tenella infection.
Parasite and chickens
The LY E. tenella strain used in this study was maintained by passages in Chinese Yellow Broiler chickens for oocysts production. Unsporulated oocysts were isolated by filtration, centrifugation and flotation. Oocysts sporulation was carried out by incubation at 29°C for 48 h in a solution of 2.5% K2Cr2O7 solution. After washing, the sporulated oocysts were counted using a cytometer prior to the inoculation.

One-day-old male Chinese Yellow Broiler chickens were obtained from Luoyang Guanghua Hatchery, China. These chickens are reared in coccidiosis-free conditions for 14 days and allowed free access to drinking water and diet without any anticoccidial drugs. The experimental protocol conformed to the guidelines of the Institutional Animal Care and Use Committee of China (No. Ykz1992.24).
Experimental treatment
A total of 80 14-day-old chickens were weighed and then divided into two groups with 40 chickens in each group. (1) The control group, the chickens were dosed with 1 mL of distilled water through oral gavage as sham inoculation but were not inoculated with E. tenella oocysts. (2) The infected group, the chickens were treated with a dose of 8 × 104 oocysts/chicken suspended in 1 mL of distilled water through oral gavage.
Histological examination
The chickens were sacrificed at the 120th hrs of treatment. The splenic tissues were collected, fixed and sectioned before hematoxylin-eosin staining. For the examination with transmission electron microscope (H-7500, Hitachi, JANPAN), the splenic tissues were fixed in 2.5% glutaraldehyde in phosphate buffer and sectioned at 50 nm before uranyl acetate and lead citrate staining as described previously (Tian et al., 2014).
Quantitative Real-time PCR
Total RNA of splenic tissue was extracted using TRIzol® solution (Invitrogen, USA). Genome were removed using RNase-free DNase I (40 U/mg RNA; Takara, Japan) prior to cDNA synthesis using transcriptase kit (Invitrogen, USA) in accordance with the manufacturer’s instructions. The specific primers of IL-2, IL-1β, IL-6, IL-10, IFN-γ, TNF-α and β-actin housekeeping gene (Table 1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. China.  As described by Liu et al., (2010), QRT-PCR was performed using a SYBR® Premix Ex Taq™ (Perfect Real Time) kit (Takara, Japan). The experiment was repeated in triplicate.

Table 1: Primer sequences with their corresponding PCR product size and position.

Immunohistochemical localisation of IgA
As our previous report (Tian et al., 2014), paraffin sections were subjected to hybridization with monoclonal mouse-anti chicken IgA (Santa Cruz Biotechnology, USA) and biotinylated goat anti-mouse IgG second antibody. Signal was detected using a DAB kit (Boster Biological Technology, Co., Ltd., Wuhan, China).
Statistical analysis
Data was analysed by Student’s t-test. Data were showed as mean±standard deviation. p<0.05 was considered to be significant differences.
Structural characteristics of the chicken spleen
The chicken spleens in the control group displayed normal appearances with regular structural organisation and considerable amounts of lymphocyte in white pulp and red pulp were observed (Fig 1. a1, a2 and a3). With the infection of E. tenella, splenic lymphocytes number decreased. The cells were arranged in disorder, the intercellular space was enlarged and some nuclei were condensed (Fig 1 b1, b2 and b3). The splenic lymphocytes ultrastructure was normal in the control group (Fig 2a), but exhibited irregular and cracked membranes, swollen mitochondria and formed vacuoles in the infected group (Fig 2b).

Fig 1: Morphological observation of the chicken spleen (400×). (a1, a2 and a3) control group, the spleen displayed normal appearances with regular structural organisation. (b1, b2 and b3) infected group, the splenic lymphocytes were arranged in disorder, the intercellular space was enlarged and some nuclei were condensed.

Fig 2:Ultrastructure of lymphocytes in the chicken spleen (5000×). (a) control group, (b) infected group. CM, cell membrane; NM, nuclear membrane; Mc, mitochondrial; IS, intercellular space.

The spleen, as the body’s largest filter, can trap and remove blood-borne pathogens and lymphocytes from circulation. The T and B-cells in the blood are attracted to their respective domains in the white pulp under the control of specific chemokines (Green et al., 2009; Saez  et al., 2011). The activation, proliferation and differentiation of these immune cells are also regulated by cytokines and chromatin dynamics (Bortnick and Murre, 2015). As such, the spleen plays a key role in protecting hosts against coccidian infections (Rose and Hesketh, 1982; Ma et al., 2012). In this study, the immune response triggered excessively by the large-scale coccidian invasion, thus, the ultrastructure of the splenic lymphocytes was altered.
mRNA expression profiles of cytokines in the chicken spleen
Compared to the control group, the mRNA expression levels of IL-2 and IL-1β in the infected group decreased by 40% (p<0.01) and 43% (p<0.05), respectively. By contrast, the mRNA levels of IL-6, INF-γ and IL-10 increased by 158% (p<0.01), 464% (p<0.05) and 379% (p<0.01), respectively (Fig 3).

Fig 3: QRT-PCR analysis of cytokines in the chicken spleen. *p<0.05, **p<0.01.

Cytokines, such as IL-2, IL-1β, IL-6, IFN-γ, TNF-α and IL-10, are major immunoregulatory proteins or glycoproteins released by activated cells; these proteins also affect the function of other immune cells (Joosten et al., 2013; Ji et al., 2016; Manjari et al., 2019). As such, cytokines are considered as molecular mediators of specific and non-specific host responses to infectious and inflammatory agents (Gabay, 2006; Thakur et al., 2019). E. tenella infection of caecum epithelial cells elicits a remarkable mucosal inflammatory response. So, the expression levels of the related cytokines in the splenic tissue were determined in this study.

IL-1β and IL-2 are multifunctional cytokines mainly secreted by activated mononuclear macrophages and T lymphocytes, respectively. These cytokines regulate antibody production and growth and/or differentiation of lymphocytes, including T, B and NK cells (Rydbirk et al., 2019). The two cytokines can synergistically improve the NK cells activity and induce release of inflammatory mediators. The downregulation of the mRNA expression of IL-1β and IL-2 is possibly related to the transfer of lymphocytes to the caecum to participate in the chicken’s immune defence mechanism controlling the intestinal inflammation induced by E. tenella invasion. IL-10, as a kind of class 2 cytokine, can downregulate the synthesis of pro-inflammatory cytokines (Kessler et al., 2017). This result of the mRNA expression of IL-10 increased by 379% (p<0.01) is an important indicator of host protection against E. tenella-induced inflammation in chickens. The IL-1β and IL-2 downregulation may be partly caused by the IL-10 expression to perform a regulatory role and to reduce immune-mediated damage. IFN-γ plays an important role in innate and adaptive immunity against bacterial, viral and protozoan infections which can eradicate intracellular organisms. In the present report, the mRNA expression of IFN-γ increased by 464% (p<0.05), which is consistent with that reported by Cornelissen et al., (2009).
IgA production in the spleen tissue
Compared with that of the control group, with the infection of E. tenella, the IgA production in the spleen tissue was decreased by 44.43% (p<0.01) (Fig 4a, 4b and 5) in the infected group.

Fig 4: IgA localisation in the chicken spleen (×400). (a) control group, (b) infected group. The arrow indicates the IgA-producing cell.

Fig 5: IgA production in the chicken spleen. ** p<0.01.

Chicken immune proteins, including IgA, are secreted to resist invading pathogens, such as E. tenella. Using immunohistochemistry techniques, we determined the IgA production in the splenic tissues. We observed that the IgA production in the spleen decreased significantly in chickens suffered from a large number of E. tenella infection. This is probably because that excessive IgA was transmitted to the caecum mucous to eliminate the pathogens. In addition, coccidia are major stress-inducing factor that cause blood loss and secretion and IgA consumption. Based on the histological and ultrasturctural observation, we suspect that the decrease in IgA production is also probably caused by the ultrastructural damage to the immunocytes in the splenic tissues. IL-6 produced by various cells participates in the terminal differentiation of B-cells and promotes the proliferation of endothelial, T and plasmablastic cells (Ramsay et al., 1994; Sato et al., 2003). In our study, the increase in IL-6 expression may be correlated with the compensatory adjustment action to the decrease in IgA production in the splenic tissue. 
These results indicated that the spleen is implicated in the antagonism of E. tenella infection. These findings suggest a strategy to control coccidiosis by improving chicken peripheral immunity.
Authors thank the China National Natural Science Foundation (Grants Nos. 31472238 and 31101855) and Young Backbone Teachers Training Project of Colleges and Universities in Henan Province, China (grant no. 2016GGJS-061) for financial support.

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