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].

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].