INTRODUCTION:

The poultry industry, especially chicken, was a fast-growing business because it had a high demand. One type of chicken that could be used as chicken poultry is layer chicken. It was inversely proportional with the common poultry knowledge. Therefore, it needs innovation published as the knowledge target to increase the production of chciken or reduce the risk of maintenance. It could be decreased the efficiency of poultry could be reduced to meet people's needs. One critical thing in maintaining chicken is the choice and supply of feed additives.¹

In the middle of 1994, S. enteritidis infection in chicken had occurred sporadically and widely reported. The infection in chicks with 7 days has a systemic quality and could be death. However, chicken of more than 14 days could be carrier chronic and cloud spread by feces each time. S. enteritidis was a foodborne bacterium from poultry that could be spread vertically (egg) and horizontally, which could be dangerous to the healthy human being.²

S. enteritidis infection in humans or animals could disturb intestinal track to gastroenteritic.³ It was reported that the prevalence of Salmonella infection in chicken meat in some cities in Indonesia was reported to be 46.6%. The annual incidence of Salmonellosis in humans in the world has been estimated at 93.8 million cases. Based on the European Food Safety Authority and the European Centre for Disease Prevention and Control report on 2004-2015, the highest Salmonella infection from chicken meat and processed product and the most extensive food-related infection from contaminated animals. The national drug and food control did research about microbiology risk quantitative Salmonella in the chicken product. 42% positive DNA isolation was obtained for Salmonella infection with the most likely number (PMA) mean <1.0 MPA/g between 0.36-2.30 MPA/g.⁴

Coffee was an essential plantation product that had better economic value than another product of the plantation. Coffee has been consumed since the 9th century in all countries.⁵ Coffee was composed of an active molecule of biology, such as phenol. Phenol was an easy molecule to find in the plant that could eat. Chlorogenic acids (CGA) were phenol-descending molecules found in Coffee and large quantities.⁶ Some of the research they have already done to learn the potency of Coffee in some of the risk diseases intensely in recent years, which showed that the component in Coffee had contributed to reaction protection of the body's reactions. The compound in Coffee that had already been used in the research was polyphenols.⁷

The progress of the poultry industry by some of the chicken infections could be affected at the global economic meeting of the chicken animal product.⁸ The efficiency enhancement of the poultry industry is not just done by increased sanitation but by giving feed additives as an immunomodulator. It was beneficial for the increased immune system of chicken to make production increase and maintain chicken quality.⁹ Some research has shown the advantage of Coffee that could stimulate bifidobacteria growth in selective and help keep the gastrointestinal health. It was for increased the efficiency of the poultry.^8,10 Therefore, it still needs to research the effect of extract coffee robusta Lampung as a feed additive in the immune system of the chicken layer.

MATERIALS AND METHODS:

The animal that was used in this research was 1^st-day strain chicken layer ISA brown and already had a value of University Brawijaya No. 1142-KEP-UB ethically.

Methode Extraction Coffee Robusta Lampung:

The Method of coffee robusta lampung extraction had done in UPT Materia Medika, Batu, Indonesia. The extract coffee robusta Lampung is 15000ml ethanol 90% and 414gram robust Lampung coffee on bottle glass. After that, cover the bottle glass and centrifuge it at 50 rpm until it is homogeneous. Filter the liquid extract that gets from the processed before and steam used Rotary evaporator. The final result is brown liquid. It will be used in this search.¹¹

Preparation of Animal Model:

Chicken must adapt to the environment by giving food and water via ad libitum. The adaptation gave the chicken freedom from fear and stress as long as the research made the appropriate environment.¹² One way to protect the chicken from stress in the research is by giving supplements on 1,5th day and 11th day. Furthermore, giving vaccine ND-IB on the 4th day and vaccine ND G7B and AI H5N1 on the 10th in the chicken also could reduce the stress.¹¹ The chicken had to be fasting 10 hours before giving by extract coffee robusta Lampung. The giving extract coffee robusta Lampung on 3-16th day as much as 0,5ml by oral. The chicken that was used in this research was chicken layer 1st-day strain ISA brown. The chicken was divided into 5 groups with 12 replication, which is healthy chicken control group (C-), group (C+) (chicken infected by S. enteritidis 10⁸CFU/ml), T1 (chicken gave 500mg/kg bw coffee extract, and infected by S. enteritidis 10⁸CFU/ ml), T2 (chicken gave 1000 mg/kg bw coffee extract and infected by S. enteritidis 10⁸CFU/ml), and T3 (chicken gave 1500mg/kg bw coffee extract and infected by S. enteritidis 10⁸CFU/ml).

The Analyzed of Relative Level CD4⁺, CD8^+, and CD45⁺ lymphocytes Cell:

On the 20^thday, the necropsy had done in the chicken and took lymph organs. Prepare mortar and PBS; after that, make suspension from lymph organs with smooth the organs used the mortar and added PBS, crushed until it becomes suspension and then used the pipette to move the suspension into Eppendorf. Then, analyzed used flow cytometry. Flow cytometry procedure: (i) prepare the conical tube, then add 50μL suspension liquid of lymph organs used pipet. (ii) add 10µL reagent antibody anti-CD4 FITC, anti-CD8 FITC, and anti-CD45 PerCP (BioLegend, USA), (iii) used vortex mixer to homogenize it, then incubate 15 minutes with temperature 20-25°C. (iv) dissolve 450µL aquades with 50µL FACS lysis liquid; after that, homogenize it. (v) after incubation finished, add 450µL FACS (lx) reagent then homogenized. (vi) incubate 15 minutes with 20-25 °C.^13,14 The sample was analyzed using a flow cytometer (BD FACSCalibur, USA).

Analysis Data:

The relative level of TCD4⁺, TCD8^+, and TCD45⁺ cells were analyzed qualitatively by ANOVA (p<0.5).

RESULTS:

The relative level of T CD4⁺ cell:

This research showed that the relative level of TCD4+ cells in chicken with cocoa Lampung as prevention bacteria S. enteritidis infection was significantly different between treatment groups. The relative level of TCD4+ cells between the normal control group and T1 and T2 group had shown not different significant but different significance with T3 group. The relative level of TCD4+ was increased in the T3 treatment group. Look at Figure 1.3.1.

Figure 1.3.1 the relative level of TCD4⁺ cell in chicken, notation a, b, and c showed different significant between-group (P<0,05).

The relative level of TCD8⁺ Cell:

Giving extract coffee in layer chicken to prevent infection of the bacteria S. enteritidis had shown the relative level of TCD8+ cell difference significantly between the treatment group. Group C+ different significant with group C-, but group C- is not different significant with all treatment groups by giving extract coffee. The relative level of TCD8+ cells is not significantly different between the T1 group with T2 and T3 group. Furthermore, the T2 group was different significant than the T3 group. Look at Figure 1.3.2.

Figure 1.3.2 The relative level of TCD8⁺ cells on chicken showed differences between the normal control and treatment groups. It was shown with notation a, b, and c (P<0,05)

The relative level of TCD45⁺ Cell:

The relative level of TCD45+ Cell on chicken that was given by extract coffee lampung as prevention of S. enteritidis bacteria infection showed different significance between treatment groups. Group C+ different significant with group C-. Group C- not different significant with T1 and T2 group, however different significant with group T3. The higher dose of extract coffee will make the relative level of TCD45+ either, look at Figure 1.3.3.

Figure 1.3.3 The relative level of TCD45⁺ Cell had show different significant between normal control group with treatment group that show by notation a, b and c (P<0,05)

DISCUSSION:

The T cell was part of the adaptive immune system. The T cell had a role in against infection such as bacteria, protozoa, fungi, and viruses. The T cell had a part called T cell memory; it had a role in recognizing specific antigens and antigens that ever infected an individual with a different level of infection or same. It was a matter to make T cells working more effectively and efficiently than Cells in the immune system nonspecific. The T cell that is activated by exposed bacteria will be differentiated into 3 subtypes which is T helper cell (CD4+), T cytotoxic cell (CD8+), T regulator/suppressor cell (CD25+, CD45+).¹⁵ Giving S. enteritidis concentration 10⁸ CFU/ml could cause the chicken to sick.³ It was caused by one of the genes that had a role in immune system response nonspecific or innate immunity. It was a Toll-Like Receptor (TLR). The nonspecific immune system was the first defense against the infectious agent and could directly respond to the antigen of the infection. One of the TLR parts is TLR4, and it could transcribe the TLR4 receptor. The ligands of the TLR4 receptor are lipopolysaccharide (LPS). It was the cell wall of gram-negative bacteria, like Salmonella sp. LPS are salmonella endotoxin bacteria that can cause inflammation when infected with the host.¹⁶

The Coffee had the green color as the natural color obtained from the acid substance with ferric chloride. One of the substance acids found abundantly in Coffee is Chlorogenic acids (CGA).¹⁷ CGA are components of Coffee that have a high bioavailability on a metabolic process in the gastrointestinal tract.¹⁸ Some studies said that CGA could be an antioxidant, a hepatoprotective, and a hypoglycaemic. ¹⁹

Some research showed that Coffee could be antibacterial, such as Streptococcus mutants bacteria and some Enterobacteria strains. It caused Coffee to be composed of Chlorogenic acid (CGA), caffeine, trigonelline, protocatechuic acid, caffeic acid, and melanoid obtained from the roasted process and have a role as antibacterial. The melanoid in Coffee, known as the antimicrobial cause, had characteristics as bacteriostatic and bactericide. The primary mechanism that melanoid did as an antimicrobial is a chelating activity that could influence bacteria's cell wall become lesions. The complexity of melanoid molecule was compound by high antioxidants produced by Maillard reaction from the combination of sugar and amino acids. ²⁰ The proinflammatory reaction caused by oxidative stress and the response to inflammation caused by bacterial infection could stimulate levels of TNFα, IL-6, and IL-1β. Some studies have already shown that the extracted Coffee was compound by antioxidants that have an anti-inflammation role. It was shown with the increase of TNFα and IL-6 levels by activity IL-10. Interleukin 10 (IL-10) is a cytokine anti-inflammatory that could hinder LPS from the induction secretion of a proinflammation cytokine such as TNFα, IL-1β, and IL-12 with blockage of NF-κB activity. ²¹

The result of relative TCD4+ Cell on chicken that treatment by extract coffee Lampung had sown that significant difference between the treatment group. The negative control group (C-) is significantly different from the positive control group (C+). T cells were immune cells that were in the body as an indicator of the system immune adaptive. T cell divided into 2 which is, T cell CD4 and T cell CD8. T cell CD4 or T helper cells, which in healthy individuals caused by T cell subsets, had a role to help B cells produce antibodies. Therefore, T CD4 cells could exist in healthy individuals.²²

Group C+ T level was increased due to S. enteritidis infection. It was stimulated macrophage as an antigen-presenting cell (APC) that could produce IL-12. APC presents bacteria peptide by MHC II and is known as T cell CD4. T cell CD4 that binding with MHC II produces IL-2. Interleukin 2 (IL-2) and IL-12 were stimulated to differentiate T cells CD4 from Th1 cells. Th1 cell produces IFN-γ. ²³ IFN-γ would stimulate macrophage activation for phagocytosis bacteria by forming reactive oxygen (ROI) and nitrite oxide (NO) that stimulated tissue damage. ²⁴

The negative control group (C-) was not different significant with T1 group and T2 group because the T1 group and T2 group was the optimal dose of extract coffee against S. enteritidis bacteria that could be in and did penetration, invasion, and multiplication inside of the chicken by increased the relative level of TCD4⁺ cell. The highest dose of extract coffee, the highest the relative level of TCD4+ cell either. The extracted coffee robusta Lampung compound by phenol molecule is known as CGA. That molecule had an antioxidant activity.²⁵ The role of CGA as an antioxidant could activate the immune system by Mitogen-Activated Protein Kinase (MAPK) pathway, which is stimulated by macrophage. Macrophage that activated will change CGA as a peptide molecule after that will bind by molecule histocompatibility complex (MHC) and presented by an antigen-presenting cell (APC). The next stage will be recognized by the T cell receptor or B cell. The peptide binding could increase the production of phospholipase C, which had a role as the enzyme stimulator protein kinase C. After that protein kinase C enzyme stimulated the production of interleukin-2 (IL-2). IL-2 induced proliferation of TCD4⁺cells and it was caused the relative level of TCD4⁺ cells to increase. In the T3 group with a dose of 1500 mg/kg bw, the ralative level of TCD4+ cells increased more than normal. T helper cell or CD4 was a Cell that could protect the stability of the immune system by regulation system. The relative level of TCD4⁺ cells that excess in the body could cause autoimmune diseases.²⁶

The relative level of TCD8⁺ cells on chicken treated with coffee robusta Lampung to prevent S. enteritidis bacteria infection was significantly different between all the groups; look at figure 1.3.2. The C+ group was different significant from C- group. On healthy or normal chicken, lymphocyte cell TCD8⁺had a role as a regulation system immune with phagocyte or terminated an immune response (Jani et al.,2011). While in the condition with bacteria infection, TCD8+ cells will activate and have a role in killing the Cell infected with bacteria by phagocytosis process. ²⁷

The C- group is not significantly different from all the treatment groups. It was caused that extract coffee robusta Lampung with 500-1500 mg/kg bw doses could prevent the infection of Salmonellosis on chicken. The extracted coffee robusta Lampung consists of CGA that, given with appropriate dose, could stimulate macrophage activation. ²² Macrophages will secrete more substances that have a role in the reaction inflammation and cell lysis mediated by T CD8 cells to protect the body from free radical damage. ²⁷

The relative level of TCD8+ cells on the T3 group was increased with no different significant response to another treatment. Activation of the TCD8+ Cell caused the immune response to stop and the uncontrolled lysis cell to stop. It was caused T CD8 cell is the T cytolytic cell, which is the killer cell and the Cell that completes the immune process response (suppressive Cell). ²⁸

Based on the result of the research had shown that ratio of the TCD4 cell and TCD8 cell on all of the groups, respectively, group C+, C-, T1, T2, and T3 is 0.4/6.2; 0.19/12.7; 0.27/13.3; 0.35/10.72 and 0.7/15.52. The relative level of the TCD8 Cell is higher than the relative level of the TCD4 Cell. In the C+ group, the activation ratio of TCD8 cells with TCD4 cells was lower than in another treatment group. This research showed that treatment with extract coffee robusta Lampung at different doses could prevent or eliminate S. enteritidis bacteria infection with stimulated activation of TCD8 cells higher than TCD4 cells. Because TCD8 cell more prevents intracellular infection than TCD4 cells. It was shown that Coffee could be an immunomodulator.²⁰ T cell receptors will stimulate the proliferation of T cells to become T CD4 cells and T CD8 cells. It will increase cytokine expression and stimulate Th1 cells such as TNFα, IFNγ, and IL-2 in the T cell. IFNγ stimulated NK cell cytolytic function and T CD8 cells.²⁹

The result of the relative level of TCD45⁺ Cell on the chicken differed significantly between the C- group with the C+ group. In the healthy condition, lymphocyte cell TCD45 had a role as regulation by decreasing the activation of the T cell receptor and MHC molecule antigen or increasing the affinity of T cell activity. It was the same as activation of the Th cell. When bacteria infect the body, the T CD4 cell will activate CD8 cell by MHC; other than that, MHC will activate the T CD45 cell to stimulate the B cell to make a memory cell and help the T cell to increase the affinity when against the antigen.³⁰

The chicken group C- is not significantly different from the treatment group that treatment by extract coffee, such as T1 group and T2 group, to prevent infection of the bacteria S. enteritidis. It was caused in the dose, the optimal dose to activate TCD45 cell as in normal conditions.²² It was shown that the CGA that is consists of extract coffee robusta Lampung could stimulate CD45. While it could increase the relative level of CD45 in the T3 treatment group. It was the exact meaning of CD 45 as a T suppressor cell, which is a Cell that could be decreased B cells and cell T helper cell (CD4) activation. T CD 45 cell was needed as an immunosuppressant in the body to prevent exaggeration of the activation cell. Based on the result of this research, it was shown that the relative level of TCD45⁺Cell was increased on the treatment group T3 than another treatment group in the C- group. Exaggerated activation of the TCD45 Cell could increase the affinity of the T CD4 cell. If the affinity of T CD4 cell increased, T CD8 cell either. It will increase against the Cell in the body and caused autoimmunity.²⁶ The result of this research had shown that the treatment group T1 and T2 with 500-1000mg/kg bw dose of extract coffee robusta Lampung could stimulate the activation of lymphocytes TCD4 Cell, TCD8 cell, and TCD45 Cell caused by S. enteritidis bacteria infection as a healthy control group (C-).

CONCLUSIONS:

This research showed that the extract coffee robusta lampung with 500-1000mg/kg bw could increase the immune cell regulation system.

COMPETING INTERESTS:

The authors have no competing interests.

ACKNOWLEDGE:

Ministry of Research and Technology Indonesia and the University of Brawijaya as research support

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Received on 04.10.2021 Modified on 14.12.2021

Research J. Pharm. and Tech 2022; 15(10):4783-4788.

DOI: 10.52711/0974-360X.2022.00803