Since mastitis is a complex infection, the effectiveness of treatment is determined by the experience of the physician, selection of effective antibiotics, timing of treatment initiation and herd health management ⁶. Sheep mastitis are commonly associated with Staphylococcus spp. Staphylococcus aureus (17-57%) and coagulase-negative Staphylococcus aureus (10-52.6%) are isolated from sheep mastitis. Also, according to the frequency of occurrence; Streptococci, Pasteurella, Enterobacteria and Corynebacteria are isolated. These agents cause clinical and subclinical mastitis. The prognosis of the disease worsens. Gangrene occurs when Staphylococcus aureus produces alpha (Hla) and beta toxins (Hlb) and other components contributing to its pathogenicity ^7,8. The most important predisposing factors of gangrenous mastitis (GM) are lack of milking hygiene and irregular vacuum settings of milking machines. In large enterprises, irritations occur in the udder tissue during the milking process and some S. aureus in the environment may cause gangrene in the udder tissue with their toxins ⁹. GM is a severe condition that frequently results in mortality ¹⁰. In bacterial mastitis, S. aureus is the most important pathogen in sheep and goat mammary glands. Mastitis caused by S. aureus can cause GM in its subclinical, chronic, acute forms ¹¹. GM is a peracute condition causing purple/black discolouration with increasing redness and oedema in the distal region of the udder. It is thought that GM may be caused by the accumulation of leukocytes in the mammary glands and the secretion of exotoxins by bacteria, as well as significant inflammation. Rainard et al. ¹² found substantial levels of Hla in the milk of goats with GM, indicating that toxin-producing strains of S. aureus are the most prevalent cause of GM in livestock and dairy ruminants. According to Anderson et al. ^13, Hla is primarily responsible for the gangrenous type of mastitis, which is caused by vasoconstriction of the arteries and ischemic necrosis. GM is extremely difficult to treat; medicines are ineffective, and if not treated promptly and effectively, it results in udder loss and death of the animal, as well as udder tissue degradation. Although Hla is the primary cause of GM in animals, beta toxin plays a role in the disease's development. The Hla causes pore development in mammalian cells, stimulating inflammatory responses. It is the most powerful membrane-damaging protein. This toxin's hemolytic, neurotoxic, and dermonecrotic qualities all contribute to its pathogenicity ^14,15. Beta toxin, also known as sphingomyelinase has significant hemolytic activity against sheep erythrocytes. It also aids bacterial colonization of the skin by destroying keratinocytes ¹⁶. Beta toxin kills secretory epithelial cells in cattle mammary glands, increasing the action of Hla. It enhances the agent's adherence to udder epithelial cells and boosts its proliferation in the area ⁸. Panton-Valentine leukocidin (PVL) is the toxin that has the greatest effect on immune cells. PVL positive S. aureus isolates that generate leukocyte toxins are commonly found in deep skin and soft tissue infections such as skin abscesses and severe necrotizing pneumonia, indicating that PVL is a significant virulence factor ¹⁷. Fibronectin-binding proteins (FnBP) binding to fibronectin/fibrinogen host cells, allowing them to spread ¹⁸.

In this study, the components isolated from 85 CMT positive clinical mastitis milk samples from two different areas were investigated in terms of antibiotic susceptibility and S. aureus virulence genes (Hla, Hlb, FnbPA, LukMF) that might cause GM. Various media were employed to naturally produce the toxin, which could then be used to make toxoid vaccines and antiserums. The hemolytic activity of the generated toxin was evaluated utilizing blood agar.

Material: In this study, a total of 85 milk samples, 79 with non gangrenous mastitis and 6 with GM, sent to Selcuk University Microbiology Department for diagnosis from Southeastern Anatolia and Central Anatolia regions were used.

Cultivation and Biochemical Identification: The samples were cultivated on 5% defibrinated sheep blood agar (Merck, Darmstadt, Germany) and Mac Conkey agar (Merck, Darmstadt, Germany) and incubated for 24-48 hours at 37 °C in both aerobic and microaerophilic conditions. Gram staining was used to identify the colonies. Catalase and coagulase tests were performed for gram-positive bacteria. Lactose, mannitol, motility and urease tests were applied to gram negatives. All S.aureus suspected strains were subjected to antibiogram test to determine resistance status (OB: Cloxaciline, N: Neomycin, APX: Ampicillin/cloxacillin, L: Lincomycine, CEQ: Cefquinome, P: Penicilline, FEP: Cefepime, SXT: Trimethoprim /sulphamethoxazole, CFP: Cefoperazone). The antibiogram test was performed using the Kirby Bauer technique. Following incubation, zone diameters were measured and analyzed using the CLSI ¹⁹. After biochemical testing, gram-positive bacteria with positive catalase and coagulase tests were selected for DNA isolation.

DNA extraction and PCR: Wizard® Genomic DNA Purification Kit was used for DNA isolation. The isolated DNAs were stored at -20°C to be used in the PCR process. The isolated DNAs were firstly screened for S. aureus spp. The DNA isolates were first confirmed for S. aureus spp. and then the positive isolates were analysed for other virulence genes. Primers and loops specific to the genes analysed were used (Table 1). For the LukF gene, the gene region with accession number S65052.1 was used and primer design was made via NCBI. PCR mix was 4 μL 5x master mix (SolisBiodyne, Estonia), 0.2 pmol of each primer was taken, 5 μL DNA was added and completed to 20 μL with ultrapure water. After PCR, they were visualised under UV light using a 100 bp marker (Solisbiodyne, Estonia) on an agarose gel containing 10 μg/mL GelRed.

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Table 1: Primers and loops specific to the genes analysed

Medium Trial for Natural Production of Toxin in Bacteria: A medium was assessed for the synthesis of Hla, the most significant toxin involved in the pathogenesis of GM. In the medium test, the strain determined to be Hla positive by PCR was inoculated in Luria Bertani (LB) peptone water, Tryptic Soy Broth (TSB), Nutrient broth, Todd Hewitt Broth (TH) and Brain Heart Broth (BHI). The strain was inoculate the medium and incubated at 37 °C for 24-48 hours. After incubation, the media were centrifuged. The supernatant of each medium was taken and subjected to the SDS-page process ^24-26.

Detection of Toxin by SDS-PAGE: For the separating gel, all ingredients are added to the solution. After adding the ammonium persulphate (APS), the liquid was poured between the glass plates, leaving a 1.5 cm space at the top. To smooth the surface of the gel, 0.3 mL of n-butanol saturated with pure water was squeezed onto the surface of the gel from the edge of the cassette. After polymerization was completed, n-butanol on the surface was poured off and washed with distilled waterIn another flask, a 5% loading gel was prepared. Again, all materials except APS were mixed in a magnetic stirrer. APS was also added and poured into the polymerized separation gel, with a comb used to produce wells in the gel. After the polymerization was finished, the comb was removed and put in an electrophoresis tank (Thermo Scientific, EC120 Mini Vertical Gel System) filled with Tris-Glycine Buffer. Samples with 4× SDS Loading Buffer and 1 M DTT were mixed with sample preparation buffer for a total of 10 min. After incubation at 95 °C, 25 μL of each sample was loaded into the wells. Electricity current was adjusted to 90 volts as the proteins moved through the 5% stacking gel and 150 volts when they reached the 12% separation gel. The electrophoresis procedure was stopped 2.5 hours after the sample reached the separation gel line.

Hemolytic Activity of Strains: After it was determined that the toxin was produced in BHI and TSB, BHI was used to determine the haemolytic activity of the strains since the most prominent band was obtained from BHI. Four Hla-positive bacteria obtained from GM samples were grown in BHI and cultured at 37°C for 24-48 hours. After incubation, it was cultivated in 100 μl of blood agar and incubated at 37°C for 24-48 hours.

Statistical Analysis: Statistical analyses were carried out using the GraphPad Prism 9.5.0 version, c, test was performed to see wether or not there is significant diferernce betwwen virulence genes of GM and non gangrenous mastitis isolates, p values of <0.05 were considered significant.

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Table 2: Antimicrobial resistance of isolated strains

PCR Findings: By culture and biochemical tests, 9 strains were found to be suspected S. aureus. A total of 9 strains were confirmed to be S. aureus by 16S rRNA in PCR analyses. Five strains were obtained from milk with suspected non gangrenous mastitis, and four were isolated from milk with GM. The virulence genes of the isolates (Table 3) were examined, and the probability of generating GM was assessed using the virulence genes caused by mastitis S. aureus isolates. Two-tailed t-test results revealed that virulence genes of strains with non-gangrenous mastitis and virulence genes of gangrenous mastitis were significantly different. Since p value was recorded as ˂0.0001 in two-tailed t-test results.

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Table 3: Virulence gene distribution of the strains

According to Table 3, only 5 of the isolates isolated from non ganrenous mastitis milk contained Hlb, LukF, and FnBA. Non-gangrenous mastitis isolates were negative for the virulence genes screened. In strains with GM, isolate 4 was found to be Hlb-negative. Figures 1 and 2 show the Hla PCR images of the isolates, which play the most critical role in developing GM.

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Figure 1: PCR image of Hla gene in GM isolates (1,2,3,4 numbered strains found positive)

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Figure 2: PCR image of Hla gene in non-GM isolates (1,2,3,4,5 numbered strains found negative) 6: Negative control, 7:Positive control M: marker

SDS-PAGE Analysis Findings: By SDS-PAGE, BHI and TSB were found to be the best media for the production of Hla (Figure 3).

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Figure 3: Hla production demonstration with SDS-PAGE. (Sample no. 3 is TSB, sample no. 8 is BH medium and these samples were found positive for Hla 33 kDa, other samples are negative)

Hemolytic Activity Findings: After incubation in BHI, the strains were cultivated in 100 μL of sheep blood agar and incubated at 37 °C for 24-48 hours. After incubation, the capacity of all strains isolated from GM milk to produce active haemolysis on blood agar was evaluated. Phenotype was shown to represent the genotypic trait.

In this study, virulence genes and antibiotic susceptibilities of the isolates obtained from mastitic milk were investigated. In addition, due to the importance of the losses caused by gangrenous mastitis in dairy sheep farms, virulence genes harboured by S. aureus isolated from mastitis strains and the potential of strains harbouring these genes to cause gangrenous mastitis were investigated. The predominance of Hla, the most significant toxin of S. aureus, which causes GM in mastitis strains, beta toxin, leucocidin F, and fibronectin-binding protein A were investigated. The virulence gene profile of gangrenous strains was compared with the virulence gene profile of non gangrenous strains.

According to the results obtained from the study; Staphylococcus aureus was detected in 15% (9/60) of 60 colonies (5 from non ganrenous mastitis and 4 from GM milk) and 85% (51/60) of other bacteria were isolated. In studies conducted with clinical mastitis milk, 13.95%, 20%, 7.6%, 9% isolated S. aureus ^29-32. When similar studies were taken into consideration, it was determined that the findings obtained in this study were close.

Antibiogram results indicate that the strains we the most resistant to Cloxacillin. According to the antibiogram results of similar studies, tetracycline, penicillin and chloramphenicol were the most resistant antibiotics ^31,33,34.

According to the PCR results from this investigation, only one of the mastitis-related isolates tested positive for beta toxin, luk F, and FnbP A. Hla, Hlb, luk F, and FnbP A were identified positively in isolates from GM. S. aureus 38% isolated from clinical and subclinical mastitis samples in sheep and goats tested positive for LukMF ³⁵. Since there were not enough studies on gangrenous mastitis in sheep, studies on goats and cattle were taken into consideration. In a study on goats with subclinical mastitis, 7.5% of the isolated S.aureus were found to be FnBA positive ³⁶. In a study conducted with milk of cattle with subclinical and clinical mastitis, Hla, Hlb, FnbA, LukF were found positive in 100% of the strains in the virulence genes sequence analysis. Better results were obtained in this study due to the difference in method and primers used ³⁷. In another study examining the virulence genes of S. aureus isolated from bovine mastitis, 83.8% FnBA, 98.7% hla, 99.1% hlb were found positive ³⁸.

The medium utilized in the toxin generation experiment included TSB, nutrient broth, TH, BHI, LB strain, and peptone water. According to the findings, experiments on manufacturing BHI and TSB had comparable outcomes. Similar to this investigation, Palmer et al. ^25, Hildebrand et al. ^24, Menzies and Kernodle ²⁶ employed TSB to produce Hla and were satisfactory.

Consequently, PCR was used in this study to detect the presence of Hla and other virulence genes that are the primary cause of gangrenous mastitis. Ampicillin/cloxacillin resistance was found in 88.8% of S. aureus isolates obtained from mastitis and GM strains. When the virulence gene profiles of the isolated mastitis-related strains were investigated, the presence of the other virulence genes tested, particularly the Hla gene, was shown to be statistically insignificant. It has been established that GM strains' hemolytic characteristics are genotypically and phenotypically compatible. According to the data obtained with the strains employed in the study and other technique components, it is believed that strains with this strain profile are unlikely to induce gangrenous mastitis.

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