Introduction

Bovine mastitis is among the most common diseases impacting dairy cows globally (1 , 2) . Visible abnormalities, such as redness, swollen udder, and fever, readily identify clinical bovine mastitis in dairy cows. The cow's milk appears watery with flakes and clots (3) .

Contrary to clinical mastitis, sub-clinical mastitis has no apparent abnormalities in the udder or milk; it is associated with less milk production and an elevation in somatic cell count (SCC). (4) .

Staphylococcus aureus is frequently associated with subclinical mastitis and causes significant financial losses due to decreased milk quantity and quality (5) . S. aureus produces several virulence factors, such as enterotoxins and leukocidins, that impart immune system resistance and facilitate adaptability. These substances help the bacterium avoid the host immune system and form an intramural infection.

Therefore, S. aureus's diversity of virulence factors significantly influences how animal infections develop. Two significant public health issues are the discovery of bacteria resistant to antibiotics for bovine mastitis and the possibility that unpasteurized dairy products could spread to humans.

Another danger in the community is antibiotic residues in milk, and the abuse of antibiotics to treat mastitis can lead to resistance (8). Since β-Lactam antibiotics have long been misused on dairy farms to treat mastitis, the rise of resistant bacteria and veterinary medications in milk poses serious public health problems. (9).

As a result, MRSA linked to livestock poses a global risk to both humans and animals. A primary reason for methicillin resistance in staphylococci is the expression of the mecA gene or its homolog mecC. It contains various staphylococcal cassette chromosomes, known as SCCmec, which are based on a mobile genetic element (10) . A trans-peptidase that is only 63% identical to PBP2a, encoded by mecA, is encoded by the novel genetic determinant known as mecC, defined as mutations in mecA.

MRSA isolates with mecA positive are seen in cattle and other animals and also spread from other Staphylococci or livestock-associated MRSA to human MRSA. (11) . For instance, cytotoxin, known as Panton-Valentine Leukocidin (PVL), encoded by the pvl gene, a virulence feature, is implicated in leucocyte destruction and necrosis of the tissues. (12) . This study aimed to identify subclinical mastitis in cows caused by Staphylococci. (in particular S. aureus). Moreover, to determine the virulence genes of MRSA among S. aureus isolates.

Material and Methods

Samples collection

Out of 200 milk samples were collected from raw milk cows from different parts of Basrah province. The sample collection started on 21 November 2023 and ended on 6 February 2024. The raw milk samples were submitted to CMT.

Microbiological techniques

Isolation and identification of bacteria: All samples were primarily submitted to the California mastitis test (CMT) and categorized by CMT scores (13). The positive CMT samples were transported immediately to the Central Research Unit in the Veterinary College using an icebox. Upon laboratory arrival, the milk sample was inoculated in Brain Heart Infusion broth (BHI) and incubated at 37°C overnight. The preincubated samples were subcultured on Mannitol Salt Agar (Himedia, India) and CHROMagar™ Staph aureus. (Chromogenic Media "pioneer/ France). The plates were incubated aerobically for 24 hours at 37°C. Gram's stain identified the suspected colonies on CHROMagar™ Staph aureus.

Molecular techniques

Genomic DNA extraction: According to the manufacturer's recommendations, a DNA kit (Geneaid, USA) was used to extract the genomic DNA of probable S. aureus isolates.

Detection of the nuc gene:Table (1) shows the primer of the nuc gene. The amplification conditions were implemented in (14).

Molecular detection of MRSA:Detecting the mecA, mecC, and pvl genes for the molecular confirmation of MRSA isolates. Table (1) Provided primer sequences. The PCR heat cycling protocol comprised based on (15).

Results

Subclinical mastitis: Ninety-five (48%) percent of the California Mastitis Test indicated positive for CMT. (table 2) illustrated the percentage according to CMT results. Figure (1). Using conventional microbiological techniques, The rate at which suspected S. aureus isolates using Mannitol Salts Agar and CHROMagar™ Staph aureus was 90 (45% ) and 70 ( 35%), respectively, as shown in Table (3). All suspected isolates were subjected to PCR using the nuc gene Table (3), Figure (2), and Figure (3). The molecular assay results were 38 (19%) characterized as S. aureus. It was determined that the difference between these outcomes was statistically significant (p< 0.05).

Figure (1). Results of California Mastitis test based on the degree of gel formation

Figure (2).A: Staphylococcus aureus appeared as golden yellow colonies on Mannitol Salts Agar,B:Chromogenic agar: colonies seemed mauve (pink), C: Gram-positive cocci.

Figure (3).Electropherogram of nuc gene amplification. The PCR product was run on 1 % agarose gel, stained with ethidium bromide. L: mean Ladder and nuc gene product size approximately (423 bp).

Detection of MRSA isolates: Methicillin-resistant S. aureus (MRSA) multiplex PCR virulence gene results for mec A and pvl genes revealed that 71% of the isolates were mecA and 16% were pvl, but none had mecC. Table (4), figure (4) A and B.

Figure (4).A:Percentage of ( mec A, mec C, and pvl ) genes in S. aureus isolates

Figure (4).B: Electropherogram of amplification of mecA,mecC, and pvl genes. The PCR products were run on a 1 % agarose gel stained with ethidium bromide. L: mean Ladder, mecA (162 bp, pvl: 85 bp ), whereas none of the isolates carried mec C.

Discussion

The dairy business suffers the most significant financial losses globally due to bovine mastitis, which also risks consumers' health (16) . However, subclinical mastitis in dairy cattle is a significant and quiet issue that causes farmers to suffer more economic losses. In addition, it leads to low milk yield and quality, considered the first disease that causes significant loss to owners (17) .

In this study, the California mastitis test results revealed that 46 % of the samples tested positive for CMT. This result agrees with (18) ., who found that the frequency of subclinical mastitis was 42.5 %. In Iraq, studies like (19) . found that 38.89% of the samples in Al Sulaimaniyah Province had subclinical mastitis. In other studies, CMT varied from 38% to 56.6% compared to other research in Basrah (20 , 21). Staphylococcus aureus (S. aureus) is considered among the most essential udder pathogens, causing significant economic losses worldwide (22) . The isolation rate of Staphylococcus aureus from SCM was 19 %. This incidence was consistent with a study conducted in Nepal that found that 15.2% of milk tested positive for CMT had S. aureus (23) . However, our study's S. aureus prevalence is more than that of a study by (24) ., in which 6 (2.8%) cows with SCM had S. aureus.

This incidence was consistent with a study conducted in Nepal that found that 15.2% of milk tested positive for CMT had S. aureus contamination (23) .. The prevalence of S. aureus is higher than what was found in a study by (24)., where 6 (2.8%) cows with SCM had S. aureus contamination.

The difference in S. aureus prevalence between this study and previous research could be explained by variations in isolation technique, geographic areas, and sample characteristics (size, season, and type).

mecA is the most significant approach to MRSA isolation (25) .. The lowest β-lactam potential altering protein (PBP2a) on SCCmec-resistant genomes is encoded by the mecA. (26) .. PCR results indicated that 71% of MRSA isolates carried the mecA gene. This rate agrees with (27) ., who reported that the mecA percentage was 86.66%. Moreover, Hammadi and Yousif announced that 88% of S. aureus cases were MRSA (28) .; in contrast, (29) observed that MRSA is found in just 10% of S. aureus infections. Meanwhile, the mecC gene was not found in any of the isolates in this study. This result agrees with (30 , 31) , who reported that none of the isolates carried the mecC gene. These findings disagree with (32) who reported that 3 (12.5%) were positive for mecC. One of S. aureus's main virulence factors is PVL. It makes it more harmful by hastening apoptosis and eliminating mononuclear and polymorphonuclear cells (33) . The occurrence of the pvl gene in MRSA isolates has not been extensively studied. According to earlier research, the pvl gene's predominance ranges from 2% to 35%. (34 , 35). Conversely, this study's findings for the pvl gene were 6 (16%), near the lower limit from other studies. Ultimately, this study showed that MRSA was common in cows with subclinical mastitis. This major public health issue should make veterinarians more knowledgeable about antibiotics.

Conflicts of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

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