Immunohistochemical and pathological changes in BALB/c mice immunized with whole sonicated Listeria monocytogenes antigens and the effect of probiotics

Article Highlight

1- The research focused on an important topic, Listeria monocytogenes as platform for vaccination.

2- The authors highlighted the possibility of using ultrasound as a prospective treatment method for animals against the disease.

3- The pathological changes of spleen appear reactive hyperplasia of lymphoid follicles especially in G3.

4- Increase number of immunopositive as focal F4/80+ immunopositive cells (brown color) compare with negative control.

Immunohistochemical and pathological changes in BALB/c mice immunized with whole sonicated Listeria monocytogenes antigens and the effect of probiotics

Thamer Jaddoa Shihab and Zainab Ismail Ibrahim

Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Tikrit, Iraq

dr.thamer.vet@tu.edu.iq, 0000-0002-7285-0157, 07811770036

zainabalrubaei@covm.uobaghdad.edu.iq, 0000-0003-1997-4465, 07703912918

Abstract

The current study was undertaken to investigate the role of macrophages as a cellular immune function against immunization with whole sonicated Listeria monocytogenes antigens (WSLMAgs) and the effect of probiotics. A preparation of WSLMAgs containing whole L. monocytogenes cell, after two subcutaneous immunization of BALB/c mice with 0.5ml WSLMAgs (0.5mg/ml) at an interval of two weeks. The bacterial identification was conducted by a conventional culture method using Listeria selective media PALCAM and confirmed by Polymerase Chain Reaction (PCR), As well as, the immunohistochemical and pathological change of it was studied in vivo by inoculating mice with pre-challenge WSLMAgs and post-challenge with virulent L. monocytogenes (1×10⁸ CFU/mL). The results revealed the cellular immune function against pre- and post-immunization in spleen organ via lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles and marker F4/80+ show the immune-positive cells in aggregation adjacent to lymphoid follicle or focal aggregation of macrophages between follicles. In conclusion, the effectiveness of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for applicants.

Keywords: Listeria monocytogenes,Antigens, probiotics, Macrophage, immuno-histochemical (IHC).

التغیرات المناعیة الکیمیائیة والمرضیةالنسیجیة فی الفئران البیضاء الممنعة بمستضدات اللیستریا المستوحدة الکاملة الصوتیة وتأثیر المعزز الحیوی

ثامر جدوع شهاب و زینب إسماعیل إبراهیم

فرع الأمراض وأمراض الدواجن، کلیة الطب البیطری، جامعة تکریت، تکریت العراق

الخلاصة

أجریت الدراسة الحالیة للتحقق من دور الخلایا البلعمیة کوظیفة مناعیة خلویة ضد التمنیع بمستضداتاللیستریا المستوحدةالکاملة الصوتیة وتأثیر المعزز الحیوی. حضر مستضد اللیستریا المستوحدة الکامل الصوتی الذی یحتوی على جمیع ترکیبات لهذه البکتریا اللیستریاالمستوحدة، وبعد التمنیع تحت الجلد لمرتین فی الفئران البیضاء بمقدار 0.05 مل من مستضد اللیستریا المستوحدة الکامل الصوتی 0,5 ملغم/مل بفاصل أسبوعین. شخصت البکتریا بالطرق التقلیدیة والزرع على الوسط الانتقائی الخاص، تم تأکیدها بتفاعل البلمرة المتسلسل، بالإضافة إلى ذلک، تمت دراسة التغیرات المناعیة الکیمیائیة والمرضیة فی الجسم الحی عن طریق تمنیع الفئران باستخدام مستضد اللیستریا المستوحدة الکامل الصوتی قبل وبعد التحدی باستخدام البکتریا الضاریة اللیستریا المستوحدة 1×⁸10 خلیة جرثومیة/مل. کشفت النتائج عن وظیفة المناعة الخلویة ضد التمنیع قبل وبعد فی عضو الطحال عن طریق فرط التنسج اللیمفاوی فی اللب الأبیض واندماج الجریبات اللمفاویة واظهر المُعَلم المناعی ان الخلایا المناعیة الموجبة فی تجمع مجاور للجریب اللمفاوی أو تجمیع بؤری للخلایا البلعمیة بین الجریبات. فی الاستنتاج، ان فعالیة اللیستریا المستوحدةالصوتیة قبل وبعد التمنیع ثم التحدی باستخدام بکتریا اللیستریا المستوحدةالضاریة فی تحریض الاستجابة المناعیة الخلویة، قد یکون بمثابة قاعدة للتمنیع مستقبلا.

Introduction

Listeriosis is a bacterial disease caused by Listeria monocytogenes that affects small ruminants (1). Listeria monocytogenes, is a Gram-positive facultative rod-shaped bacterium, causes listeriosis, a serious and life-threatening illness caused mostly by ingesting contaminated foods (2), and is a food-borne intracellular pathogen that causes listeriosis also is widespread in the world (3). Listeriosis is characterized through a wide-ranging variety of diseases divided into two types, includes severe invasive listeriosis and non-invasive febrile gastroenteritis (4).

Listeria monocytogenes was utilized as a model organism in our investigations on the immunogenicity of lethally sonicated bacteria, As a result, Listeria monocytogenes serves as a rigorous model for evaluating lethal sonicated would retain bacterial immunogenicity and result in a superior vaccine when compared to other ways of pathogen death (5), In addition, the use of a sonicator in this study might have a significant influence on albino mice's cellular immunity (6). L. monocytogenes is a prototypic inducer of the cellular adaptive responses, whose antigen-specific effectors are cytotoxic CD8+ T cells (7). Furthermore, due to the unique process of listeriolysin O pore formation and listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and multiply rapidly (8). The administration of prophylactic probiotics could be an important treatment option for preventing infectious complications and modulating immunity (9), also, the probiotics decrease the severity of clinical signs and increase the immunity respond that can be used as medicinal and remedial method (10). + present in many local tissue macrophages, comprising the red pulp macrophages in the spleen, microglia in the brain, Kupffer cells in the liver, and Langerhans cells in the skin, are strongly and constitutively expressed (11). The efficiency of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for humans and livestock applicants.

Materials and methods

Animal

Adult females BALB/c mice (n=30) were purchased at 6-8 weeks of age from the Animal House/College of Veterinary Medicine/Tikrit University/Iraq, and kept in specific cages at the same animal house at a room temperature of 22±3 °C with a 12-hour light/dark cycle, with food and water supplied ad libitum. All experiments were carried out in accordance with Baghdad University's Institutional Animal Care and Use Committee regulations. All animals received humane care. For this study, the mice were divided into three groups at random, ten mice in each groups, 1^st. Group (control positive) challenge with virulent strain L. monocytogenes only (1x10⁸CFU/ml); 2^nd. Group Immunized with 0.5ml WSLMAgs (0.5mg/ml) S/C for each mouse in 1^st. day and repeated after 2 weeks of immunization; 3^rd. Group immunized with both WSLMAgs as in 2^nd. group and administration oral probiotics (Vitane Pharmaceuticals, Inc USA) (0.02 mg/ml) for each mouse, twice in week for 4 weeks. At 28 day from start experimental half animal of each grouped euthanized for histopathological and immunohistochemistry (IHC) investigations immune response change in spleen (GA), and half of the remains animal of each group were challenged with virulent L. monocytogenes 1 × 10⁸ CFU/ml, After three day of challenge, all mice were euthanized and samples were collected for histopathology and IHC for immune response change in spleen (GB).

Bacteria

The bacterial sample was achieved from Media Diagnostic Center in Erbil, Iraq. virulent Listeria monocytogenes were commonly cultivated at 37 °C on Brain heart infusion agar for stimulation of bacteria and after activation on it transported to Listeria Identification Agar Base (PALCAM) (HiMedia, India) M1064 with Listeria Selective Supplement (PALCAM) (HiMedia, India) to avoid mutation of bacteria also as selective identification of L. monocytogenes by providing a grey-green with a black center and a black halo (12).

Polymerase Chain Reaction (PCR)

According to ([i]) to check strain in ASCo. lab Baghdad/ Iraq, the primer 27F, 5`-AGAGTTTGATCCTGGCTCAG-3`; and 1492R, 5`-TACGGTTACCTTGTTACGACTT-3` at Annealing Temp. (60 ^oC), finally Size (bp) 1500 bp.

Preparation of WSLMAgs from Listeria monocytogenes according to (14).

The subsequent has been prepared as follow Listeria monocytogenes were cultured on Brain Heart Infusion Agar with a 24-hour incubation period at 37°C. For 30 minutes, the precipitate was centrifuged at 3000 rpm at 4 °C and extracted with PBS 7.2, at that time washed three times with PBS, resuspended with PBS, and introduced into the universal tube. The universal tube containing Listeria monocytogenes suspension was placed in an ultrasonicator (Karl Klob – Germany) at 12 Peak pulse/sec at 2 minute intervals for 30 minutes in a cool atmosphere (ice). For 30 minutes at 25000 rpm, the sonicated suspension was centrifuged. The supernatant fluid was verified through gram stain and culture on blood agar to establish the antigen's sterility.

The total protein concentration of this antigen, which was measured using the Biuret method 9.1 mg/ml then it was watered-down to become 0.5 mg/ml this antigen was considered as Whole Sonicated Listeria monocytogenes Antigens (WSLMAgs). In order to prepare the whole soluble Sonicated Listeria monocytogenes antigens (WSLMAg), to remove cell debris, the homogenate was centrifuged twice at 14000 rpm for 30 minutes each time. The supernatants went through the 0.22μm Millipore filter and were preserved at-20C previously they were used. The watered-down fluid was examined by gram staining and cultured on nutrient agar and Listeria Identification Agar Base (PALCAM) to verify the antigen's sterility. According to Biuret technique, the total protein concentration of this antigen was 0.5 mg/ml. These antigens were considered to have destroyed soluble whole sonicated Listeria monocytogenes antigens (WSLMAgs).

Histopathological study

The organs obtained from each mouse were subjected to the following steps (15). The organs fixed directly in Neutral buffered formalin 10%, dehydrated, and embedded in paraffin for storage. Then, 6-mm sections were cut, de-paraffinized, and rehydrated using a standard protocol.

Immunohistochemistry (IHC) staining

The antibodies used for surface staining included anti- F4/80 antibody (Fine Test); Catalog No.: FNab02922 and Rabbit-DAB(Poly-HRP) Detection IHC Kit (Fine Test) Catalogue No.: IHC0007. It entails using the idea of antibodies (Abs) attaching particularly to antigens in biological tissues to detect antigens (proteins) in cells of a tissue section (16). IHC was performed on de-paraffinized and rehydrated sections. Briefly, slides were incubated with antigen retrieval solution for 10 minutes. Slides were then incubated in serum block for 60 minutes, followed by rat anti-F4/80 Ab for 60 minutes. Endogenous peroxidase activity was blocked using 3% H₂O₂. Cells were subsequently incubated with a Rabbit-DAB (Poly-HRP) Detection IHC Kit, and all slides were counterstained with hematoxylin.

Results

Polymerase Chain Reaction (PCR)

The molecular results of the 16S bacterial rRNA region showed a positive amplification of 1500 bp amplicon for the Listeria monocytogenes bacteria when use sequencing and analyzed with GenBank databases.

Table 1: Shows percentage the immunopositive cells of macrophages (F4/80+) antibody Pre- and post-immunization then challenge with Listeria monocytogenes in slide microscope of experimental groups

Virulent Listeria monocytogenes (Positive control G1B)

In post-challenge G1B group, the prominent microscopic lesions in tissue sections of spleen were infiltration of mononuclear cells mainly lymphocytes and macrophages, with lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles with histiocytic proliferation (Figures 1 and 2).Also, slimly increase percentage of the immunopositive cells in Table 1 for IHC staining (45.77) in red pulp of spleen also differ than other groups (Figure 3).

Figure 1: Histopathological section of spleen in post-G1: Shows follicular hyperplasia in white pulp (arrow) and coalescence of lymphoid follicles (arrow). (H&E stain, 40X).

Figure 2: Histopathological section of spleen in post-G1: Shows lymphoblast’s proliferation in the center of hyperplastic lymphoid follicle (arrow) and remnant of cellular debris (arrow). (H&E stain, 400X).

Figure 3: Immunohistological section of spleen in post-G1: Shows histiocytes F4/80+ immunopositive cells (brown color) peripheral to eccentric arteriole (arrow). (DAB Chromogen staining, 200X).

Pre Immunization with WSLMAgs G2A and then post Challenged with Virulent L. monocytogenes G2B

In pre-challenge G2A group, the tissue sections of spleen represented with moderate histiocytosis in red pulp as dilated vascular sinuses and contained mononuclear cells with proliferation of megakaryocytes and hyperplasia of lymphoid follicles (Figure 4). Furthermore, In post-challenge G2B group, The spleen presented reactive hyperplasia in white pulp, also proliferation and infiltration of histiocytes mainly mature (basophilic) lymphocytes noted as dark one uniforms and regular rounded cells (Figure 5). Moreover, in post-challenge G2B the percentage of the immunopositive cells for IHC staining in red pulp (65.01) (Figure 7) higher than the pre-challenge G2A (62.86) and G1B (45.77) Figure 6 and Table 1.

Figure 4: Histopathological section of spleen in pre-G1A: Shows congestion of vascular sinuses in red pulp (arrow) ; and filled with histiocytes (arrow) with megakaryocyte (arrow). (H&E stain, 200X).

Figure 5: Histopathological section in spleen of post-G2B: shows lymphocytic proliferation (light center) (arrow) in white pulp. (H&E stain, 200X).

Figure 6: Immunohistological section of spleen in pre-G2A: Shows focal and diffuse immunopositive cells (M1/ F4/80+) (arrow). (DAB Chromogen staining, 200X).

Figure 7: Immunohistological section of the spleen in post-G1B: Shows immunopositive cells (F4/80+/brown color) in peripheral follicular tissue and in red pulp (arrow). (DAB Chromogen staining, 100X).

Immunized with WSLMAgs and oral administration of probiotic (Pre G3A and Post G3B challenge):

In pre-challenge G3A group, in spleen shows reactive hyperplasia of lymphoid follicles in white pulp of spleen and moderate thickening capsule with histiocytosis (Figure 8). Moreover, in post-challenge G3B, marked by subcapsular dilation and filled with histiocytes with Malpighian corpuscle (Figure 9, 10). Besides, in pre-challenge G3A the percentage of the immunopositive cells for IHC staining 65.56 in red pulp (Figure 11) lower from the post-challenge G3B (66.66) (Figure 12).

Figure 8: Histopathological section in spleen of pre-G3A: Shows moderate thickening fibrous capsule (arrow) and marked histiocytosis mainly lymphocyte (arrow) in red pulp. (H&E stain, 200X).

Figure 9: Histopathological section in spleen of post-G3B: shows subcapsular dilation (arrow) and filled with histiocytes and reactive hyperplasia of lymphoid follicles (arrow) in white pulp. (H&E stain, 100X).

Figure 10: Histopathological section in spleen of post-G3B: shows hyperplasia of malpighian corpuscle (arrow) in white pulp with megakaryocyte (arrow). (H&E stain, 200X).

Figure 11: Immunohistological section of spleen in pre-G3A: Shows focal and peri-follicular infiltration of F4/80+ immune-positive cells (brown color) (arrow). (DAB Chromogen staining, 500X).

Figure 12: Immunohistological section of spleen in post-G3B: Shows focal F4/80+ immunopositive cells (brown color) in red pulp and near lymphoid follicle (arrow). (DAB Chromogen staining, 100X).

Figure 13: Immunohistological section of mouse spleen paraffin-embedded slide using (F4/80+ Antibody) (DAB Chromogen staining, 500X).

Discussion

The results of the post-challenge G1B group agreed with Yin (17) who described that the virulent strain induced a strong immune response substantial pathological changes in the liver and spleen due to is Listeriolysin O responsible for the development of a pore forming external toxin named LLO, which is necessary for vacuole membrane lysis and cytoplasmic release of Listeria monocytogenes, Also, Phosphatidylinositol-specific phospholipase C (PI-PLC) and zinc-dependent broad-spectrum phospholipase C (PC-PLC) are encoded by plcA and plcB, respectively, which facilitate the escape of the pathogen from single in addition double-membrane bound vacuoles synergistically with LLO Kyoui (18), and the results of IHC for G1B agreed with Kohyama (19) who reported presence of F4/80+ in red pulp of spleen due to infiltration of macrophage. Additionally, The results of Group 2 agreed with Awni (20) who revealed that the antigens promote cellular immunity in immunized mice and infiltration of innate and adaptive cells in spleen; also, WSLMAgs antigen can trigger both innate and adaptive immune responses, and because WSLMAgs contains all Listeria monocytogenes structures that can stimulate innate immune cells, phagocytic cells to secrete pro-inflammatory cytokines such as TNF-α and IL-12, which play an important role in initiating acquired immune responses Al-Bayati (14), in addition to stimulated Toll-like receptors on dendritic cells (DCs) elicited protective T cell responses in mice Datta (5). The IHC of WSLMAg group agreed with Warschkau (21) who told F4/80+ may have a role in the generation of IFN-γ and IL-12 by macrophage cells once they are exposed to WSLMAgs similar to heat-killed Listeria monocytogenes (HKL) when infection with facultative intracellular pathogens such as LM requires IFN-γ to limit bacterial growth and which activate NK cells for IFN-γ release and macrophages were triggered once more. Also, The results of Group 3 agreement with Yap (22) who reported that the combined effects of Lactic Acid Bacteria's cell wall structure (polysaccharide) immunomodulatory characteristics and WSLMAgs might generate a stronger immune response at lower vaccination dose and give more effective protection against infection; Besides, increased CD4+ and CD8+ T cells, NK cells and CD8+ DC, and play an important function in Listeria protection through the capacity to produce IL-12 and trigger IFN-γ producing CD4+ and CD8+ T cells, additionally, the CD8α+ DCs are essential in the presentation of Listeria antigens to defensive CD4+ and CD8+ T cells Mitchell (23). Moreover, accepted with Kearney (24) who noticed large lymphoid aggregation in spleen involved many lymphocyte follicles packed with B and T lymphocyte, B cell proliferation, precursors of plasma cells that production antibody during immune reaction. In addition, The IHC of Group 3 agreed with Dubyak (25) who reported that different forms of G protein-coupled receptors (GPCRs) play a crucial role in host organisms' immune responses to infection or damage to sterile tissue. Also, microbe-induced macrophage/NK cell contact involves direct cell-to-cell communication and provides the first evidence for the functional significance of the macrophage surface glycoprotein F4/80+ Dubyak (25).

Conclusion

Immunization with WSLMAgs was efficient and provides protection against bacterial infection by induction cellular immunity pre and post immunization, might serve as an immunization platform for applicants.

Conflict of interest

We declare that no conflict of interest present with any other published papers.

Acknowledgments

Thanks are extended to the College of Veterinary Medicine, University of Tikrit, for facilitating the work in the college laboratories.