SteE regulation of Th1/Th2 cytokines expression in chickens during S. Pullorum infection

. Nowadays, timely monitoring of zoonotic agents, including salmonellosis, which are caused by various serovars of the family Salmonella, is relevant. Attention should be paid to the study of cytokine levels in combination with immunological studies. This helps clarify the pathogenesis of infectious diseases and develop preventive measures. The main purpose of study was to detect the process of regulating Th1/Th2 cytokines expression in chickens infected with salmonellosis. The field strain of S. Pullorum CVCC 530 was used in the research. The steE deletion mutant (Δ steE ) and steE -complemented Δ steE : steE (Δ steE + steE ) strains were constructed in the WT strain using the λ -Red recombination method. Chickens were orally infected with WT, Δ steE , and Δ steE + steE strains (1×10 9 CFU/individual). The effect of steE on the host immune response remains unknown. Compared with the group infected with the WT or Δ steE + steE strain, IL-12 and IFN-γ mRNA transcript levels were significantly higher, while IL-10 mRNA expression was significantly reduced in the liver and bursa infected with the Δ steE strain; IL-4 showed a dramatically reduced transcription level, but IL-18 mRNA expression was significantly increased in the Δ steE strain – spleen, cecum, and heart; IL-10 mRNA expression was significantly reduced in the spleen and cecum infected with the Δ steE strain. These results suggest that steE may regulate the Th1/Th2 cytokine response balance in chickens infected with S. Pullorum and provide new insights into the pathogenesis of salmonellosis for the treatment of persistent infection


Introduction
Poultry breeding is widely developed throughout the world.The concentration of a large number of poultry in a limited area creates favourable conditions for the emergence and spread of infectious diseases among the livestock of poultry farms.Salmonellosis, which is accompanied by damage to the gastrointestinal tract and septicaemia, is one of the poultry diseases that causes concern among veterinary medicine specialists.According to L. Zhike et al. (2021), salmonellosis agents pose a threat to human health because salmonellosis is a zoonotic disease.
Infection of poultry with salmonellosis can occur both from the parent flock and during the cultivation of commercial broiler stock.In most cases, given the relatively short life span of broilers, S. pullorum infection in most cases occurs vertically from the parent flock.Infection of poultry with the causative agent of salmonellosis can occur through the alimentary route, as well as aerogenic and transovarian (through the egg).T.I.Fotina & T.V. Sergeychik (2022) proved that an important point for the occurrence of the disease is the presence of sources of infection.Poultry, rodents, other farm animals, feed, wild animals, poor cleaning and disinfection, disposal of poultry, and workers and visitors can be infected.
Researchers substantiated that the key point in preventing the development of salmonellosis is strict compliance with veterinary and sanitary requirements and rules in poultry farms.Researchers also indicate that the disease worsens in the presence of stress factors (Zhike et al., 2021).B. Alosaimi (2020) draws attention to the fact that cytokines, which are produced by almost all cells of the body for the purpose of intercellular interaction and regulation of biochemical processes, are an important factor influencing the immune reactions of poultry.Cytokine imbalance is important in the pathogenesis of infectious diseases.
Ukrainian Journal of Veterinary Sciences.2023.Vol.14, No. 3 H.C. Webster (2022) proved that the important properties of cytokines are: pleiotropic action, the presence of overlapping and doubling phenomena in a single regulatory network.Induction conditions are created, that is, when one cytokine acts on others and regulates the expression of cytokine receptors.
Cytokines regulate specific immune reactions and the immune response to the pathogenic properties of pathogens of infectious diseases, including salmonellosis.
In this regard, the development of methods of prevention of poultry salmonellosis is urgent.The purpose of the study is the experimental reproduction of salmonellosis in chickens and the investigation of the process of regulating the expression of Тh1/Тh2 cytokines under the conditions of infection of chickens with the field strain S. Pullorum CVCC 530 (WT) using a deletion mutant.At the second stage, the immune response of chickens was investigated.In connection with the urgency of the problem and the solution of the set goal, the tasks of research were to study the effect of cytokines Тh1/Тh2 on the development of inflammatory processes in the spleen, liver, tissue bag and small intestine of chickens, under the conditions of their infection with the causative agent of salmonellosis.

Literature Review
Researchers P. (2020) state that in the normal host immune system, Th1/ Th2 cells do not differentiate, but the high expression of inflammatory cytokines causes disorder in case of Salmonella infection.X. Kang et al. (2022) demonstrated that some host-adapted Salmonella serovars can suppress the innate immune response and create an infection-friendly environment for long-term survival in host cells, such as S. Pullorum.SteE has been reported to be closely associated with colonisation of bacterial invasion in mice (Jaslow et al., 2018).Previous study (Liu et al., 2018) showed that steE was associated with persistent infection and virulence of S. Pullorum Among them, steE promotes intracellular replication of Salmonella in host cells.However, the role of steE prior to S. Pullorum infection on host immune responses remained largely unknown.In this regard, S. Pullorum-infected chickens were used as an in vivo model to investigate the effect of steE on the host's immune response.

Materials and Methods
The study was carried out during 2022-2023 at the China Veterinary Culture Collection, Beijing, China, and the Laboratory of Innovative Technologies, Safety and Quality of Livestock Products of Sumy National Agrarian University, Ukraine.

Bacterial strains
The wild-type strain of S. Pullorum CVCC 530 (WT) was obtained from the China Veterinary Culture Collection, Beijing, China.The steE deletion mutant (ΔsteE) and steE complemented ΔsteE:steE (ΔsteE+steE) strains were constructed in WT strain by using the λ-Red recombination method, and stored in laboratory (Liu et al., 2018).All strains were grown overnight in Luria-Bertani medium at 37°C with shaking prior to use in chicken infection.

Chicken infection assay
40 Hy-Line white chicks (2-day-old) were obtained from the Henan Animal Incubation Centre (China).This study was approved by the Animal Care and Ethics Committee of Henan Institute of Science and Technology.All animal studies were conducted in accordance with Directive 2010/63/ EU of the European Parliament and of the Council "On the Protection of Animals Used for Scientific Purposes" (2010) approved by the Commission on Ethics and Bioethics of the Faculty of Veterinary Medicine of the Sumy National Agrarian University (protocol No. 3 dated 12.21.2021).The chicks were randomly divided into four groups (n=10).The chicks in experimental groups were orally infected with WT, ΔsteE and ΔsteE+steE strains (1×10 9 CFU/individual), and normal control group were orally infected with PBS as previously method and dose described (Liu et al., 2018).At 3 days post-infection (dpi), the liver, spleen, bursa, cecum, and heart tissues of each chicken from each group was collected and stored at -80°C for further analysis.

Statistical analysis
All results are presented as mean ± standard deviation (SD) for at least three independent experiments.Quantitative data was analysed by one-way ANOVA using GraphPad Prism software suite, version 8.0 (Graph Pad Software Inc., San Diego, CA, USA).Statistical significance was expressed as * P<0.05 and ** P<0.01.

Results and Discussion
The study has found that SteE changes the mRNA of inflammatory cytokines levels in spleen of infected chickens by S. Pullorum.At the first stage of research, SteE level was evaluated.
It was found to alter mRNA of inflammatory cytokines levels in spleen of chickens during S. Pullorum infection.The mRNA levels of IL-18, IL-12 were much higher in spleen of the ΔsteE group than that in the WT or ΔsteE+steE strain group at 3 dpi (Fig. 1).Regarding the mRNA anti-inflammatory cytokines levels, the IL-10, IL-4, were substantially decreased in the spleen of ΔsteE group compared to the WT or ΔsteE+steE groups, whereas the mRNA IFN-γ level was not significantly different.were much higher in liver of the ΔsteE group than that in the WT or ΔsteE+steE at 3 dpi, but the mRNA level of IL-18 had no significant difference (Fig. 2).The mRNA level of IL-10 was much lower in liver of the ΔsteE group than in the WT or ΔsteE+steE groups, whereas the mRNA level of IL-4 had no significant difference.SteE changes the mRNA inflammatory cytokines levels in bursa of chickens infected with S. Pullorum.Authors were given the task of investigating SteE changes mRNA inflammatory cytokines levels in bursa of chickens infected with S. Pullorum.The mRNA levels of IFN-γ, IL-12 were much higher, but no difference was detected in IL-4, IL-18 in bursa of the ΔsteE group than in the WT group or ΔsteE+steE at 3 dpi (Fig. 3).The mRNA level of IL-10 was much lower in bursa of the ΔsteE group than in the WT or ΔsteE+steE groups.SteE changes the mRNA of inflammatory cytokines levels in cecum of chickens infected with S. Pullorum.Experimental studies of the fourth stage proved that SteE changes mRNA of inflammatory cytokines levels in cecum in chickens infected with S. Pullorum.The mRNA level of IL-18 was much higher, but no difference was detected in IFN-γ, IL-12 in cecum of the ΔsteE group than in the WT or ΔsteE+steE groups at 3 dpi (Fig. 4).The mRNA levels of IL-10, IL-4 were much lower in cecum of the ΔsteE group than in the WT or ΔsteE+steE groups.SteE changes the mRNA of inflammatory cytokines levels in heart of chickens with S. Pullorum infection.The mRNA level of IL-18 was higher, but no difference was detected in IFN-γ, IL-12, IL-10 in heart of the ΔsteE group than in the WT or ΔsteE+steE groups at 3 dpi (Fig. 5).The mRNA level of IL-4 was much lower in hearts of the ΔsteE group than in the WT or ΔsteE+steE groups.Salmonella is an intracellular pathogen that causes great harm to human and livestock health worldwide, with complex and diverse antigenicity and serotypes.Y. Hu et al. (2019) demonstrated that among common serotypes, S. Pullorum causes systemic lethal disease with high mortality in chickens within 2-3 weeks.An infected adult chicken exhibits abnormalities of the reproductive tract without serious clinical symptoms, leading to chronic or recessive infection.S. Pullorum causes significant economic losses to chicken farms worldwide, especially in developing countries.Therefore, T.I.Fotina & T.V. Sergeychik (2022) state that it is very important to control the spread of S. Pullorum in poultry farms.
S. Pullorum can spread horizontally and vertically to progeny, which is unlikely to be eliminated.Thus, accurate and rapid pathogen diagnosis is important for the control and eradication of S. Pullorum.At present, the detection of pathogenic bacteria depends on culture methods and biochemical identification, which Studies by Z. Lin et al. (2017) found that Salmonella can colonise the intestine and spleen and directly accept macrophages as target cells.After infection with S. Pullorum, the bacterium can not only escape the destruction of intracellular active substances, but also proliferate and spread within macrophages.SteE is required for Salmonella replication and virulence in macrophages.Thus, it was hypothesised that the pathogenic mechanism of S. Pullorum infection in chickens may be the same as in HD-11 cells.
In this study, steE was selected as a research gene based on the λ-Red recombination system to construct the S. Pullorum ΔsteE strain.The results showed that the growth and biochemical characteristics of S. Pullorum and S. Pullorum ΔsteE strains are similar, which is When determining the virulence, it was found that the deletion of steE had an effect on reducing the pathogenicity of S. Pullorum in chickens, and this proved the important role of steE in the virulence of S. Pullorum.In addition, the presence of bacterial colonies in the organs of chickens showed that the general trend of change in S. Sullorum is similar to that of the ΔsteE strain.The presence of colonies of WT strains and ΔsteE strains in the spleen, caecum, and bursa of chickens reached its maximum peak on day 3, and the number of WT strains and strains ΔsteE in the liver of chickens reached its maximum peak on day 4.During the process of infection, the number of ΔsteE in defferent organs of the initial stage of infection after Salmonella infection in chickens that first increased, reached the peak at the middle infection stage, and then decreased at the later infection stage.The amount of ΔsteE strains in organs of chicken was always much lower than of WT strain.And, the amount of colonisation of the ΔsteE strains in organs of chicken was always lower than of the WT strains.The results of the study indicate that steE can promote S. Pullorum colonisation in organs of chicken and contribute to S. Pullorum virulence.
Recent studies have shown that deletion of SPI-2 reduces the virulence of Salmonella, which has been attributed to the fact that SPI-2 is a DNA fragment acquired externally during Salmonella evolution.I. Panagi et al. (2020) reported that deletion of steE significantly attenuated mouse spleen colonisation and reduced S. Typhimurium virulence.SteE has been considered as an important effector in host organs for persistent Salmonella infection.SteE was encoded by the Salmonella prophage Gifsy-1, which was reported to activate the signal transducer and activator of transcription 3 signalling pathway and then produce the anti-inflammatory cytokine IL-10, thus promoting Salmonella replication in cells and increasing bacterial colonisation in vivo.S.L. Jaslow et al. (2018) identified many factors that affect the accuracy of the final results.On the one hand, the interval between organ collections was relatively long, and the exact time of penetration of bacteria into different organs cannot be precisely determined.However, only by reducing the interval between organ harvests it is possible determine the time at which bacteria settle in chicken organs to reach a peak.In addition, there were other factors affecting the amount of S. Pullorum in the testing process, such as equipment, reagents, cleanliness of the Petri dish, and errors caused by the operation, etc.
Inflammatory cytokines are one of the most important regulators of host-cell interaction in acute systemic disease (Alosaimi et al., 2020).After S. Pullorum infection in chickens, the cells of the host will secrete Th1/Th2 cytokines, which regulate the balance of the internal environment and resist the damage of external harmful substances (Wu et al., 2018).SteE Ukrainian Journal of Veterinary Sciences.2023.Vol.14, No. 3 increased the bacterial virulence, translocated into the cytoplasm of the host cell through T3SS-2, and caused the severe disseminated infection of other extra-intestinal tissues, such as the spleen and liver in mouse infected with Salmonella (Pham et al., 2020).Here, it was found that steE positively regulated the balance of Th1/Th2-related cytokines in tissues of chicken infected with S. Pullorum.
Salmonella as an intracellular pathogen, can regulate host immune response via T cell activation (Cerny & Holden, 2019).IL-12 is a pro-inflammatory cytokine that plays an important role in the host's defence against intracellular pathogen infection (Yang et al., 2018).IFN-γ production by Th1 cells and initiated by IL-12 and IL-18, induced the oxidative effect and improved the ability to suppress intracellular bacteria growth (Withanage et al., 2005).A previous study showed that sspH2 significantly decreased the mRNA expression of Th1-related cytokines (IL-12 and IFN-γ) in tissues of mice infected with S. Enteritidis (Shappo et al., 2020).Furthermore, S. Pullorum decreased the mRNA expression levels of pro-inflammation cytokine (IL-12, IL-18, and IFN-γ) compared with S. Enteritidis in infected macrophages (Tang et al., 2018).
In the current study, steE increased the Th1-related cytokines expression (IL-12, IL-18, and IFN-γ) in the chicken tissues infected with S. Pullorum, which was consistent with the previous report.SteE might be involved in Th1-mediated tissue injury and elimination of pathogen during S. Pullorum infection.Anti-inflammatory cytokines are mainly related to clear pathogens and tissue repair after Salmonella infection.L-10, as an important anti-inflammatory medium, can inhibit the expression of various pro-inflammatory factors induced by Th1 cell, and also participate in Th2 cell-mediated anti-inflammatory response (Rasquinha et al., 2021).Recent studies have found that steE can induce the production of anti-inflammatory cytokine IL-10 in the spleen of mouse infected with Salmonella, but it has no relevant data on other tissues (Jaslow et al., 2018).Moreover, the persistent infection of Mycobacterium tuberculosis needs the participation of IL-10, which indicates that IL-10 contribute to longterm systemic infection of intracellular bacteria (Park et al., 2021).Another study found that the persistent infection of Salmonella needs the support of IL-10, which is associated with the Th1/Th2 balance (Liu et al., 2018).Induced the expression of IL-10 contributed to limit the Th1 cytokines and escape the immune response of host cells ( Webster et al., 2022).In the present study, steE obviously increased the IL-10 mRNA transcript level during the infection phase.Therefore, steE may be more conducive to the repair of damaged tissues by promoting the anti-inflammatory or Th2 cytokines expression during early stages of infection.
The imbalance of Th1/Th2 cytokine is an important indicator of pathogens invasion (Abebe, 2019).Recent studies show that S. Pullorum was inclined to regulate host immunity toward a Th2-like immune response in poultry (Tang et al., 2018).In this study, results demonstrated that steE inhibited the Th1 immune response, but promoted the Th2 immune response.Furthermore, steE activity promoted anti-inflammatory M2 phenotype and facilitated Salmonella invasion into host cells, leading to increased pathogen persistence (Jaslow et al., 2018).SteE have the function of immune response of transforming different types, which is beneficial to intracellular survival of Salmonella (Panagi et al., 2020).The previous study showed that steE can also increase the virulence of S. Pullorum and aggravate the outcome of host infection (Liu et al., 2018).Thus, researchers speculate that steE-inhibited inflammatory response could be related to M2 phenotype, which was beneficial to evade immunity and persistent infection of S. Pullorum.However, it needs to be further confirmed how steE regulates the cytokine balance of Th1/Th2 in S. Pullorum-infected chicken.It has been established that the results obtained coincide with the findings of other researchers and confirm the influence of steE regulation of Th1/Th2 cytokine expression in chickens infected with S. Pullorum.

Conclusions
It was established that IL-12 and IL-18 mRNA levels were much higher in spleen of the ΔsteE group than in group of WT or ΔsteE+steE strains at 3 dpi.Regarding the mRNA anti-inflammatory cytokines levels IL-4, IL-10 were strong reduced in spleen of the ΔsteE compared with the WT or ΔsteE+steE groups, while the mRNA level of IFN-γ had no significant difference.
It was noted that IL-12, IFN-γ mRNA levels were much higher in liver of the ΔsteE group than in the WT or ΔsteE+steE groups at 3 dpi, but the IL-18 mRNA level had no significant difference.The mRNA level of IL-10 was much lower in liver of ΔsteE group than of the WT or ΔsteE+steE groups, and the mRNA level of IL-4 had no significant difference.
It was determined that IL-18 mRNA level was much higher, but no difference was found in IL-12, IFN-γ in bursa of ΔsteE group than in WT or ΔsteE+steE groups at 3 dpi.IL-10, IL-4 mRNA levels were much lower in bursa of ΔsteE group than in WT or ΔsteE+steE groups.It is argued that IL-18 mRNA level was much higher, but no difference was found in IL-12, IFN-γ in cecum of ΔsteE group than in WT or ΔsteE+steE groups at 3 dpi.IL-4, IL-10 mRNA levels were much lower in cecum of ΔsteE group than in WT or ΔsteE+steE groups.Тhe mRNA IL-18 level was much higher, but no difference was detected in IL-12, IFN-γ, IL-10 in hearts of ΔsteE group than in the WT or ΔsteE+steE groups at 3 dpi.The mRNA level of IL-4 was much lower in the hearts of ΔsteE group than in the WT or ΔsteE+steE groups.
It has been shown that steE can modulate the Th1/Th2 immune response in S. Pullorum-infected chickens.And steE enhanced the anti-inflammatory response induced by a strong Th2 immune response.Overall, this study extends to further elucidating the S. Pullorum and the host immune response interaction, providing new insights into Salmonella pathogenesis for the treatment of persistent Salmonella infection, without the use of antibacterial drugs, which will provide the opportunity to obtain high-quality and safe poultry products.

Figure 1 .
Figure 1.SteE changes the mRNA inflammatory cytokines levels in spleen of chickens infected with S. Pullorum Source: developed by the authors

Figure 2 .
Figure 2. SteE changes the mRNA inflammatory cytokines levels in liver of chickens infected with S. Pullorum Source: developed by the authors

Figure 3 .
Figure 3. SteE changes the mRNA of inflammatory cytokines levels in bursa of chickens infected with S. Pullorum Source: developed by the authors

Figure 4 .
Figure 4. SteE changes the mRNA of inflammatory cytokines levels in cecum of chickens infected with S. Pullorum Source: developed by the authors

Figure 5 .
Figure 5. SteE changes the mRNA of inflammatory cytokines levels in heart of chickens infected with S. Pullorum Source: developed by the authors are time-consuming, have a long detection cycle and low sensitivity, and cannot meet the needs of social development.Thus, Zh.Liu et al. (2018) suggest the need to develop an accurate and simple detection method for S. Pullorum serotype diagnosis.