INTRODUCTION:

Colibacillosis is an infectious disease caused by the primary agent of Escherichia coli (E. coli). E. coli is a gram-negative bacterium, rod-shaped that does not form spores, is not acid-resistant, and measures 2−3 x 0.6μm^1,2. E. coli act as primary agents through antigens and the production of enterotoxins that cause disease. Moreover, E. coli secretes toxins and performed several diseases in humans, such as gastroenteritis, diarrhea, hemolytic uremic syndrome (HUS), and kidney failure. While in poultry, colicepttisemia is found along with other abnormalities involved in pericarditis, hepatitis, folliculitis, spleen hyperplasia, and peritonitis^3–7.

The pathogenic level of E. coli is affected by the attachment ability to the epithelium of the respiratory organs and the resistance to phagocytosis of the body's immune system. There are three important antigen structures in the classification of E. coli: O antigen (Somatic), K antigen (Capsel), and H antigen (Flagella)^7,8. The active/determinant side of the O antigen is located in the oligosaccharide part, is heat resistant, and in its grouping is numbered 1,2,3, and so on. The K antigens respond in heat resistance and interfere with agglutination, while the H antigens contain proteins present in the thermostable flagella. There are 173 serotype groups of O antigens, 74 types of K antigens, and 53 types of H antigen^9–12. One of the organs that responds to the immune system is the spleen.

Spleen is an organ located on the left side of the stomach, and consisted of the cranial and abdominal parts. This organ serves as a filter for blood and coordinates an organism's immune response. In mice, the spleen is formed from mesenchyme in the dorsal part of the mesogastrium. The spleen is divided into two parts: the white and red parts. The white part (alba pulp) protects the immune system against infection, while the red part (Rubra pulp) removes unnecessary materials from the blood, such as damaged red blood cells^7,8,11,13,14. Several immune boosters like drugs and vitamins were used to enhance animal immunity. However, long-time drug consumption might be caused heart failure or kidney complications^6,9,10,12,15. Several researches previously reported that herbal medicines, including Cleome rutidosperma, Styrax officinalis, and Eryngium creticum showed antibacterial activities^16–18. Oregano leaves, an alternative herbal medicine, have several properties to remedy fever, diarrhea, nausea, jaundice, and itching on the skin. Ethanolic extract of oregano leaf also inhibits Pseudomonas aeruginosa and Staphylococcus epidermidis^19–24. However, the information of oregano’s potential activity as a growth inhibitor of E.coli at colibacillosis mice models is limited. Therefore, this research investigated the effect of various oregano extract concentrations on the spleen of colibacillosis mice (Mus musculus) Balb/C models.

MATERIALS AND METHODS:

Animal Models:

Animals were obtained from the Biomedical Laboratory of the University of Muhammadiyah Malang with a Certificate of Pathogen Free (Salmonella). Twenty mice Balb-C strain, male, aged 3–4 weeks, bodyweight of 20–25grams were used in this study. Mice were divided into five groups, which were negative control (normal mice), positive control (mice was infected with E.coli), T1 (colibacillosis + 5mg/Kg BW of oregano extract), T2 (colibacillosis + 10mg/Kg BW of oregano extract), and T3 (colibacillosis + 20mg/Kg BW of oregano extract). Colibacillosis mice models were prepared by inducing mice with 20mg/Kg BW of streptomycin through oral administration. This was done in order to eradicate the normal gut bacteria of the mice. After 24 hours of induction, mice were infected by E. coli 3 x 10⁹ CFU/mL. Positive colibacillosis was identified by the onset of diarrhea, low appetite, and inactive mice¹⁴. All of the procedures in this study was approved by animal ethics from an ethic committee of Universitas Brawijaya with approval number 095-KEP-UB-2021

Colibacillosis mice were administered oregano leaves extract orally for 3 days. Mice were euthanatized by dislocating the cervical area so that the organs were not damaged. The mice were then placed in a dorsal position, and the four extremities were fixed with needles. The mice were cleaved in the abdominal cavity to take out the spleen. The spleen was soaked into 10% formalin solution for histopathological examination²⁵.

Origanum vulgare extract preparation:

A total of 500 grams of dried oregano leaves were put in a sealed container and soaked with 96% ethanol in a shaker machine at 50 rpm for 24 hours. The homogenates were filtered with a filter cloth and evaporated using a rotary evaporator at 70ºC. The extraction was repeated three times^22,23^.

Histopathology Spleen Analysis:

The spleen was washed with NaCl and fixated with a 10% formalin for 24 hours. Then, the organ was trimmed and inserted into the cassette. The cassette was soaked into aquadest for one minute and dehydrated with increasing concentration of alcohol (from 50-95%) for 15 minutes. The spleen was embedded with liquid paraffin at 56-62 °C for 15 minutes and then was sliced using a microtome with 6 μm thickness. A sliced spleen was fixed at object glass and deparaffinized using xylol with increasing concentration. Then, the sliced spleen was rehydrated with alcohol in decreaseing concentration from 100%, 90%, 80%, and 70% for 5 minutes each. The preparat was stained using hematoxylin-eosin with standard procedure²⁵. Histopathology of mice spleen was observed using a microscope (Olympus) with 100 – 400X magnification. The histomorphometry was measured using image J application. The abnormalities of spleen histology were scored with some criteria, included no specific abnormalities (0), edema, or hemorrhage in the mild stage (1), mild stage depletion (2), moderate depletion (3), and severe depletion or hemorphination (4)²⁰.

Data Analysis:

Histopathological data were analyzed descriptively to characterize the white pulp profile each groups. The white pulp diameters were depicted as average ± standard error. Mann Whitney with p<0.05 was used to compare the significant levels of each groups.

DISCUSSION:

Colibacillosis is an infectious disease that affects the digestive organs and is caused by the bacterium E. coli. Mice induced with E. coli at 3 x 10⁹ CFU/mL dose and administered oregano extract have clinical symptoms that appear to be weight loss, lethargy, and diarrhea^{3,12,13,15,26}. The pathogenesis mechanism of E. coli begins with colonization at a certain point in the surface cells of the intestine (mucous cells), followed by cell division, destruction of intestinal cells, crossing of intestinal cells into the bloodstream, propagation to the target organ, and resulting in organ damage^3,4. Transmission from E. coli occur through drinking water and food contamination or one infected cage. Colibacillosis has clinical symptoms of decreased appetite, diarrhea, and lethargic animal^5–7.

Histopathological profile of colibacillosis mice spleen:

Histopathological profile of colibacillosis mice showed moderate depletion or hyperplation, while oregano extract administration reduced depletion and significantly improved cell to normal (Table 1). Normal mice performed a normal spleen profile and revealed any spleen histology abnormalities. Mann Whitney's test showed that the score of T1 and T2 were same indicating 5mg/kg BW oregano extract performed similar action with T2 with 10mg/kg BW of oregano leaves extract. Administration of oregano leaves extract with 20mg/Kg BW showed edema or hemorrhage in the mild stage.

Table 2. Spleen Histopathology Score

Treatment	Score
K- (Negative Control. normal mice)	0^a
K+ (Positive Control. infected with E.coli)	3^b
T1 5 mg /kg BW of oregano extracts	2^b
T2 10 mg /kg BW of oregano extracts	2^c
T3 20 mg /kg BW of oregano extracts	1^c

* Different notations show significant results (p<0.05)

Normal spleen was shown by unchanged shape and there was no germinal center. Enlarged germinal center indicated unhealthy mice, and the immune system was stimulated due to infection^2,8,9. In mice infected with E. coli, there was a lot of hyperplation in the spleen cells at a moderate level^b. Hyperplasia was an active event of the immune system due to the entry of bacteria or foreign bodies into the body. Spleen hyperplasia (splenomegaly) was one of the physiological responses of the immune system against bacteria. Infection with microorganisms, heat, radiation carcinogens, and enzymes can cause reactions in the form of spleen hyperplasia^2,8–12,15. The spleen may change due to pathogenic infections, but more noticeable changes are due to inflammatory products. Splenomegaly was caused by pathogenic organisms².

After giving oregano leaf extract, T1 treatment mice given 5mg/kg BW oregano extract showed an average spleen score of 2^b and the depletion of lymphoid cells at a mild stage compared to positive control. Lymphoid depletion was also found in the mice group given oregano leaf extract at 10mg/kg BW dose. Spleen depletion is characterized by reducing the number of lymphocyte cells in lymphoid follicles^27,28. Depletion of lymphoid tissue is an acute inflammatory response due to the migration of heterophils and lymphocytes from the lymphoid organ to the inflammatory site due to the stimulation of inflammatory mediators^9,15,29.

In the mice group with a dose of oregano extract of 20 mg/kg BW, the observation score on the spleen organ is 1^c. Blood is found around the spleen cells due to the rupture of blood vessels and depletion of lymphoid cells at a mild stage (Figure 1). The compounds carvacrol and thymol act as antibacterial, antioxidants, and anti-inflammatory in fighting free radicals caused by E. Coli ^30,31. Thymol destroyed the cell membrane in bacteria so that intracellular bacteria leak out. Carvacrol increased the permeability of bacterial cells and lysed bacterial cell membrane^30,31.

Effect of Oregano Extract on Animals: colibacillosis model:

Physiological changes in the spleen organs at the time of bacterial infection were observed through changes in the size of white pulp in the group of mice infected with bacteria. In comparison to normal mice, the infected mice showed enlarged white pulp. Positive control and T1 groups showed a wide diameter of white pulp. Interestingly, T2 and T3 revealed that oregano extract administration 10 and 20mg/Kg BW improved the white pulp diameter, close to normal mice.

White pulp is a lymphoid nodule or collection of lymphocyte cells encircling the central arteries, particularly T-11 lymphocytes^29,32. The white pulp of the T1 mice group with oregano extract therapy on 5mg/kg BW dose appeared to be 346.176μm width. This cell has the largest diameter because the infection due to E. coli activates the immune system^2,25. The spleen contains many macrophages and is the site of the creation of active lymphocyte cells and antibodies. Lymphocyte cells in germinal centers that play a role in specific immunity assemble and proliferate^29,33,34. E. coli infection triggered releasing pro-inflammatory and inflammatory cytokine leading to the congestion of spleen tissues and decreasing white pulp size in oregano treatment group caused by oregano bioactivity in inflammation. Carvacrol and thymol were reported to have antibacterial and anti-inflammatory activities. Besides that. those compounds promoted cell membrane lysis and degraded bacterial DNA^19,21.

Higher doses of oregano leaf extract in the T3 mice group resulted in a decrease in the size of the white pulp. It is known that the diameter of the pulp reaches 259.044 μm. Oregano contained some bioactive compounds involved flavonoids, phenolics, and steroid compounds that performed anti-malaria, antioxidant, antibacterial, and toxic activities^19,21,23,35. The content of antibacterial compounds from oregano leaf extract will reduce the number of pathogenic bacteria that enter the body effectively. Carvacrol inhibits the mobility of E. coli by interfering with the proton motive force. Proton motive force processes on mitochondrial membranes produced ATP that was initiated by protons. Proton disrupting inhibited flagellum movement and reduced the pathogenicity of E. coli^36–39. Thymol was an antibacterial effectivity which degraded both gram-negative and gram-positive bacteria⁴⁰.

The spleen contains many macrophage cells and is the site of activated lymphocytes and antibodies. The spleen plays a role in the body's defense against microorganisms, abnormal cell foreign particles, and in secreting old or normal erythrocytes. The spleen is a vital organ where immune cells interact with foreign microbes and is a highly effective blood filter³⁴. The antimicrobial activity of oregano leaf extract makes incoming pathogenic bacteria lose the ability to infect and replicate before entering the blood vessels of the gastrointestinal tract. A decrease in the number of pathogenic bacteria in the blood will prevent the body's excessive immune response, infection, and even systemic disorders that can be more fatal^36,41,42.

CONCLUSION:

Administration of oregano leaf extract (Oreganum vulgare) improved tissue damage shrinkage of white pulp size. Therefore. oregano extract might have potential drug activity.

CONFLICTS OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGE:

The authors would like to thank Brawijaya University. Faculty of Veterinary Medicine for their kind support

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Received on 12.03.2022 Modified on 18.06.2022

Research J. Pharm. and Tech 2023; 16(1):236-240.

DOI: 10.52711/0974-360X.2023.00043

Groups	Diameter White Pulp (µm)
Groups	N1	N2	N3	N4	N5	x̄
Negative control	366.21	260.76	235.21	301.28	252.49	283.19
Positive control	350.02	376.51	467.04	294.64	136.45	324.932
T1	208.06	333.41	333.24	470.8	385.37	346.176
T2	257.88	246.5	227.84	255.62	361.09	269.786
T3	283.03	215.62	323.26	195.2	278.11	259.044