Abstract

This study aimed to prepare and evaluate the immunogenic activity of three distinct antigens derived from S. pseudintermedius, a prominent isolate identified from otitis externa samples of dogs and human infections. The isolation and identification of Staphylococcus species were conducted using rigorous cultural, morphological, and biochemical methods, with confirmation via VITEK technique. A total of 100 samples, comprising 50 from humans and 50 from dogs, were collected. Following successful antigen preparation, twenty rabbits were immunized and divided into four groups, including a control group. Cellular immunity was assessed through a delayed hypersensitivity (DTH) skin test, revealing positive skin reactions among the immunized groups. Subsequent challenge dosing resulted in clinical observation, with marked signs of illness and severe infection in the control group. Histopathological examination further confirmed this infection and bacterial isolation. Remarkably, the prepared antigens exhibited substantial protein concentrations and induced a robust immune response in immunized rabbits against experimental L. monocytogenes infection. These findings underscore the potential of S. pseudintermedius antigens for developing protective strategies against bacterial infections in both veterinary and human medicine, offering significant implications for global health practitioners and researchers in the field.

Highlights :

• Rigorous identification: S. pseudintermedius derived antigens evaluated after precise Staphylococcus species identification using cultural, morphological, and biochemical methods, confirmed via VITEK technique.
• Robust immune response: Immunization with prepared antigens induced positive delayed hypersensitivity skin reactions and protected against severe L. monocytogenes infection, demonstrating potential for cross-species bacterial infection prevention.
• Clinical and histopathological insights: Control group exhibited illness and Listeriosis upon challenge, validated through histopathological examination, highlighting antigen-induced protection against bacterial invasion.

Introduction

Staphylococcus are tiny gram-positive cocci they got their name because they looked like bunches of grapes, currently, more than 40 known species of Staphylococcus exist [1],[2],[3].

Due to their ubiquity and adaptability, it inhabits the skin, skin glands and mucous membranes of people, other mammals and bird, as well as the environment [4] Mammals can develop localized and systemic infections from staphylococci ranging from small wounds infections to potentially fatal illness including endocarditis and osteomyelitis [5],[6],[7].

Staphylococcuse spp. can cause many sever disease in animals for example in a cattle, sheep and goat the main infection is mastitis.in dog and cat may cause otitis and skin infections. in laboratory animals specially rabbits and ginea pigs Staphylococci may cause severe pneumonia [8],[9],[10].

Many researchers had used different subunit antigens of bacteria. This protocol was initially conducted in 2002 in annual meeting preceeding that conclude that twenty-one centuries must be the century of using sub unite particles of bacteria in all bacterial vaccines because using of sub unite vaccine is safer for human and animalsand most all bacterial parts can give an immune response even with killed bacteria [11],[12],[13].

Method

Samples of dogs and humans were collected in Baghdad governorate in November 2021 to march 2022 fifty canine samples, all from otitis-infected animals, The same time frame saw the collection of human samples from 50 cases with Tonsillitis, rhinoSinusitis, and skin lesions.

Prepration of S. pseudintermedius Antigens

3.6.1 Prepration of Whole S. pseudintermedius Sonicated Antigen ( WSAg ):

The whole sonicated S. pseudintermedius antigen was done according to [14],[15].

Preparation of cell wall and peptidoglycan

The cell wall and peptidoglycan antigen of S. speudintermedius were extracted according to [16].

Experimental design

1To identify Staphylococcus samples collected from 50 human samples with tonsillitis, rhinosinositis and skin lesions and 50 dog samples with otitis. Samples were inoculated into brain heart infusion broth (BHIB), then cultured on mannitol salt agar. Bacterial identification was done depending on colony morphology, gram stain, biochemical test (catalase, coagulase,) and VITEK. Extraction of antigens was done using the most important and most prominent isolate of Staphylococci these antigens were whole sonicated antigen, peptidoglycan and cell wall antigen. After extraction, the concentration of protein in each extracted antigen was estimated using Biuret method, Doses of immunization of laboratory rabbits were estimated according to [17]. ListeriamonocytogenesIsolate used in this study obtained from the unit of zoonotic Diseases Baghdad University College of veterinary medicine, and was confirmed by vitek system. the Challenge dose of Listeriamonocytogenes 1×10⁷CFU/ml accorfing to [18] Culture L. monocytogeneswas prepared on brain heart infusion broth at 37°C for 24 hrs, and centrifuged in centrifuge at 3000 rpm for 20, the sediment washed three times with phosphate buffer solution PBS (pH 7.2) and re-suspended with 1 ml of PBS, in tube. The concentrations of bacteria in diluents tubes had compared to stander Macfarlane tube,

Experimental laboratory animals

Twenty healthy rabbit's local breed, and all rabbits from male sexes with 1500-2000 gm weight. The rabbits were housed in laboratory animal house of college of veterinary medicine they were housed in cages as stainless-steel wire with fully ventilated room at temperature 25. the animals were provided with food and water and adapted for two weeks before initiating of experiment. The animals were divided into four groups each consist of five animals and treated as follow:

First group: immunized with whole S. pseudintermediussonicated antigen S/C with (protein 32mg/ml) concentration as primary dose followed by booster dose after 14 days.

Second group: immunized with peptidoglycan of S. pseudintermediusS/C (protein 25 mg/ml) concentration as primary dose followed by booster dose after 14 days.

Third group: immunized with cell wall of S. pseudintermediusl S/C (protein 35 mg/ml) concentration as primary dose followed by booster dose after 14 days.

Fourth group: served as control was injected S/C with 1ml of Phosphate buffer solution as primary dose followed by booster dose after 14 days.

At 21-day post first immunization, Delayed Type Hypersensitivity Skin Test (DTH) was done to all groups. and at 28 days post first dose of immunization the rabbits of all groups were challenged orally with (1ml) of bacterial suspension containing 1×10⁷CFU/ml of viable virulent ListeriaMonocytogens [18]. At 35 day post primary dose of immunization the rabbits of immunized groups were sacrified then post-mortem examination was done to all sacrified rabbits.

Delayed Type Hypersensitivity Skin Test (DTH)

This test was done at 21 day post primary dose of immunization and carried out according to [19] .one side flank region of all animals of immunized and control groups was clipping and shaving and intradermal injection in the center of this region was done with 0.1ml of whole sonicated antigen of S. pseudintermedius using insulin syringe.

The results of the test were recorded by measure erythema diameter and thickness of skin at region of injection after 24 and 48 hours after injection and using vernia for this purpose.

Macroscopic examination

After 7 days post challenge dose the Animals were euthanized using closed ether jar. All surface body was accurately observed for the presence of external lesions. Then the carcasses were opened by longitudinal abdominal incision all internal organs were accurately observed to indicate any lesions or congestion. Smears were taken from internal organs for culturing to indicate the presence of L. monocytogens.

Histopathological examination

At 35 day post primary immunization, the sample was collected from vital organs of experimental animals were collected which included; lung liver, intestine, spleen and kidneys. The tissues were fixed in %10 buffer formaldehyde solution immediately after removal, after 72 hrs of fixation the specimen were washed with tap water and then processing was routinely done with assest of routinely with set of upgrading alcoholic concentration from %70 to absolute 100% for 2 hrs in each concentration to remove water from the tissues,then the clearance was done by xylol,the the specimens were soaked with semi-liquid paraffin wax two stages.Then blocks of the specimens were made with paraffin wax and sectioned by rotary microtome at 5mm for each tissue sample. All tissues were stained with Hematoxlin and Eosin (H&E) stain and the histopathological changes were observed under light microscope [20].

Results and Discussion

These antigens were used to immunize three groups of rabbits as was designed in experimental design. The three groups showed cellular immune response. this was detected by skin testing (delayed type hyper sensitivity) and histopathological changes after post mortem.

4.2.3 Delayed Type Hypersensetivity – skin test

The results of this test in the three immunized groups in addition to control group were represented by mean of skin erythema and induration and showed in table (4.7), (4.8) and figure (4.9). The skin test results indicate the presence of cellular immune response to the antigen[21].

The reaction in this test depends on the presence of memory Tcell for CD4 and CD8 [22] also the Delayed Type hyper sensitivity test depends on both T helper cells (TH1) drive responses and cell recruitment and chemotaxis to the site of injection. [23]. After antigen injected intrademally it is taken up by Langerhans cells and these migrate to the draining lymphe node in which the antigen presenting to memory Tcells that respond by generating Th1 effector cells.the Th1cells recognize antigen when they encounter it in the skin and accumulate around the antigen. [24]. the Th1 cells are responsible in secreating averiety of cytokines that recruite and activate macrophage and other nonspecific inflammatory cells[25].

The induration occure due to accumulation of activated macrophages and another inflammatory cells in the area of injection and the erythema or the red appearance of skin occure due to increase the permeability of blood vessels due to infiltration of the inflammatory cells.[26]

The variation in the diameter of erythema indicates different degree of immune response [27] the higher response may related to higher specifity that lead to high skin reaction [28]

G ₁ : immunized with whole S.pseudintermedius Sonicated Antigen

G ₂ : immunized with Peptidoglycan

G ₃ : immunized with Cell wall

G ₄ : control group (not immunized with Antigen)

Figure 1.positive reaction of skin test in one animal of one immunized group

Clinical signs observation and bacterial isolation

Signes like decrease or loss apetite with clear lost of condition and depression appeared on all the animals of control group after giving the challenge dose. after 48 of challenge dose one animal of control group was died and after 5 days another animal in the same group was died. pathological investigation to these 2 animals was done immediately after death.

Bacterial isolation was done firstly from dead animals directly after death. the other animals were authenized after 7 days of challenge and the bacterial isolation was also done.

L.monocytogeneswere isolated from all animals of control group that died and authenized and no bacterial isolation was shown among the animals of immunized groups

Pathological examination

Gross Examination

The gross examination was done after 7 days of challenge with Listeriamonocytogens. grossly control positive group show sever and generalized congestion throughout the carcass of animals of this group. Also enlarged liver with white spots were seen, with engorged gall bladder. mucous inside intestine also seen with engorgement of blood vessels of small intestine wall. These pathological gross lesions were illustrated in Figure (2) (3)

Figure 2.Gross appearance in control positive group shows enlarged liver with congestion and presence of white spots and engorged gall bladder

Figure 3.Gross appearance in control positive group show engorgement of mesentric blood vessels in small intsetine

Animals of the three groups that immunized show almost normal carcass with apparently low or no congestion with normal internal organs appearance. No significant gross lesions were seen. These were illustrated in Figures (4) (5).

Figure 4.Gross appearance in the lung of one animal of immunized group, shows no gross pathological changes

Figure 5.Gross appearance in kidneys of one animal of immunized group, shows no gross pathological changes

The gross pathological lesions in control group that reported in this study were also recorded by [29] in mice inoculated experimentally with ListeriaMonocytogens.

4.3.2 Histopathological Examination

Histopathological picture of control group show severs pathological changes throughout the organs examined. Congestion was the main picture especially in liver Figure (6) (7) due to proliferation of inflammatory cells especially neutrophil and mononuclear cells with dilatation of blood vessels and accumulation of mononuclear cells around blood vessels figure (8).

In intestinal wall section there is focal destruction in the epithelial cells of crypts figure (9). In lung there is thickening of internal alveolar septa due to accumulation of inflammatory cells figure (10) and congested capillary blood vessels. In spleen sever depletion of white pulp due to necrosis of lymphocytic cells figure (11).

Kidney section show congestion of blood vessels with sever vacuolar degeneration of epithelial lining cells of renal tubules and excessive inflammatory cells figure (12) and hyaline cast in the lumen of renal tubules that suffering from vacuolardegeneration.

All the above histological lesion indicates generalized septicemic infection and this was described by[30] as a feature of listeria infection. Infiltration of mononuclear cells in liver and depletion of white pulp of spleen were confirmed an important histopathological picture of listeria infection by [29].

Alterations of kidney section that reported by our study such as hyaline cast and signs of nephritis with the signs enteritis in intestinal wall section were noticed in mice infected with virulent Listeriamonocytogens by [31]

The immunized groups showed histological changes in internal organs included generally congestion ,infiltration of mononuclear cells and phagocytic or other inflammatory cells .the haveir mononuclear cell infiltration was in the lamina propria of small intestine of immunized groups this may related to the infection with Listeriamonocytogenesorally and that immunized groups had the ability to induce cellular immune response that produce these cells to migrate and localized in lamina propria to eliminate infection Figure(13) (14).

The highest response was in group one and two and this may indicate the ability of whole sonicated antigen and peptidoglycan to induce the higher immunity [24] Liver of group one have also higher infiltration of mononuclear cells in and fibrosis of wall of hyperplastic epithelial lining bile duct figure (15).The aggregation of inflammatory cells developed formation of granulomatous inflammation which consist of a huge number of macrophages (Histiocytes) this was obvious in group two and three figure(16) (17).

This changes in liver indicate the presence of cellular immunity and the large number of these cells in liver may be due to the nature of the function of liver in nutritional metabolic balance and elimination of toxins, body west product, metabolic substances and foreign materials including pathogen [32]. these lesionsare the characteristics feature of granulomatous inflammation that generated by a fusion of activated macrophages [33]. In lung the thickening inters alveolar septa due to mononuclear cells infiltration in group one figure (19) and hyperplasia of bronchial associated lymphoid tissue in wall of airways figure (18) indicate the high immune responseandthe ability of whole sonicted antigen as adefending mechanism against pathogen [34] while in lung of group two there is proliferation of alveolar macrophages in alveolar space figure (20) this also indicate the immunogenic response in this group.

In kidney of group one there is aggregation of neutrophil and in dilated congested blood vessels figure(21) while in group two moderate vacular degeneration in epithelial lining cells was shown figure (22) the overall picture of presence of mononuclear cells in other organs indicate the production of proinflammatory mediators such as IL₁₂ [35].The infiltration of inflammatory cells in three immunized groups may related to the active production of tumor necrotic alfa (TNF- ) which is proximal mediator of neutrophil chemotactic factor[36] and also related to that neutrophils infact is the first cell for defence .In spleen apoptosis in the white pulp resulting in the formation of spaces with cellular depris) in group one figure(23) and hyper plasiaof white pulp in group two figure (24) may related to the massive inflammation of phagocyted due to immune responses [37].

In group three marked proliferationof lymphocyte in periarteria sheath (figure (25) indicate the presense of immunoproliferative microenvironment which related to obvious immune response [38]

Figure 6.Histopathological section in the liver of animal in control positive group; shows congested blood vessels and inflammatory cells particularly neutrophila and mononuclear cells infiltration in liver parenchyma (H & E stain 400X)

Figure 7.Histopathological Section in the liver of control positive group ; shows neutrophils in dilated congested blood vessels and sinusoids in addition to mononuclear cells around bile duct in portal area (H & E stain 400X)

Figure 8.Histopathological Section in the liver of control positive group; shows neutrophils in dilated congested blood vessels and mononuclear cells around bile duct in portal area (H & E stain 400X)

Figure 9.Histopathological section in the intestine of control positive group; showess focal destruction of epithelial cells in crypts (H & E stain 100X)

Figure 10.Histopathological Section in the lung of control positive group; shows increase thickness of interalveolar septa due inflammator cells infiltration and congested capillary blood veseeles (H&E stain 400X)

Figure 11.Histopathological Section in the spleen of animal of control positive group; shows severe depletion of white pulp due to necrosis of lymphocytic cells (H & E stain 400X)

Figure 12.Histopathological Section in the kidney of control positive group shows hyaline cast in the lumen of renal tubules suffering from marked vacuolar degeneration (H & E stain 400X)

Figure 13.Histopathological Section in the intestine of animal in G1 (immunized with WSAg); Shows mononuclear cells infiltration in the lamina properia (H&Estain 400)

Figure 14.Histopathological chamges in the intestine of G2(immunized with PGN); shows marked mononuclear cells infiltration in the dilated lamina propria (H&E stain 400X).

Figure 15.Histological Section in the liver of G1(immunized with WSAg); shows mononuclear cells infiltration in fibrosis in wall of hyperplastic epithelial lining of bile duct (H & E stain 400X).

Figure 16.Histopathological Section in the liver of animal in G2 (immunized with PGN); shows granulomatous inflammation in the liver parenchyma (H & E stain 400X).

Figure 17.Histopathological Section in the liver of G3 (immunized with cell wall Ag.) e; shows multiple granulomatous lesions (H & E stain 400X).

Figure 18.Histopathological Section in the lung of animal in G1 (immunized with WSAg); shows increase thickness of interalveolar septa due mononuclear cells infiltration (H & E stain 400X).

Figure 19.Histopathological Section in the lung of G1(immunized with WSAg); shows moderate hyperplasia of bronchial associated lymphoid tissues in wall of airways (H & E stain 400X).

Figure 20.Histopathological Section in the lung of animal in G2 (immunized with PGN); shows proliferation of alveolar macrophages in alveolar spaces (H & E stain 400X).

Figure 21.Histopathological Section in the kidney of G1(immunized with WSAg);shows neutrophils in dilated congested blood vessels with acute cellular degeneration of epithelial cells lining renal tubules (H&E stain 400X).

Figure 22.Histopathological Section in the kidney of in G2 (immunized with PGN); shows moderate vacuolar degeneration of epithelial lining cells of renal tubules (H & E stain 400X).

Figure 23.Histopathological Section in the spleen of G1 (immunized with WSAg); shows focal apoptosis in white pulp left spaces contain cellular debris (H & E stain 400X)

Figure 24.Histopathological Section in the spleen of G2 (immunized with PGN); shows moderate hyperplasia white pulp (H & E stain 400X).

Figure 25.Histopathological Section in the spleen of animal inG3 (immunized with cell wall Ag.); shows marked proliferation of lymphocytes in periarterial sheath (H & stain 400X).

Conclusions

The three antigens that extracted from Staphylococcus Pseudintermedius contains good concentrations of protein. The three antigens gave an acceptable immune response in Immunized rabbits against Listeria mionocytogenes experimental infection.

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