Introduction

Mastitis is considered a primary distributed disease in dairy herds and it is a frequent infection in dairy cows and ruminants. Mastitis causes a significant economic loss via direct and indirect costs (1). The direct costs of bovine mastitis are veterinary treatment costs and require more of the labor requirements (2). The indirect cost of clinical and subclinical bovine mastitis is the significant economic losses are reduced milk yield and quality due to mastitis (3). More than 100 types of bacteria have been detected in the mammary glands of cows, but a small number of these microorganisms cause mastitis (4). The etiology microorganisms of bovine mastitis are divided into two groups: the first group is a contagious bacterium, and the second group is an environmental bacterium according to their reservoir, source, and mode of transmission (5). Contagious or environmental bacteria infect the mammary gland because of the transmission of this bacteria from the source of contamination such as a contaminated milking machine, towels, the hands of milkers, bedding, soil, and the feces from the udder of cattle (6). Successful programs are applied to control mastitis based on the cleaning and disinfecting of all the machines, and utensils that direct contact with the surface of the teats and udder (5). Staphylococcus is frequently present in various habitats such as humans, animals, and plants (6). Also, it is considered a highly significant cause that triggers clinical and primarily subclinical mastitis in cattle herds (7). Furthermore, S. aureus is considered an essential food-borne pathogen, which is mainly responsible for the cause of food poisoning cases, and outbreaks worldwide (8).The infected cow may be a primary reservoir and source of contamination of the raw milk with S. aureus (9). S. aureus possesses more than 30 different virulence factors (10). S. aureus can produce different types of exotoxins (11). Contaminated milk and dairy products with S. aureus are seen as the primary sources to trigger food poisoning to consumers (12). S. aureus can resist various types of antibiotics and disinfectants (13). This variation is based on the geographical territories, the genetic characteristics of the isolates, and the type of samples (14). Moreover, the S. aureus isolates showed resistance to all types of β-lactam antibiotics. However, yet are applied to cure of bovine mastitis (15). Methicillin-resistant Staphylococcus aureus (MRSA) was documented in the 1970s and isolated from bovine mastitis (16). MRSA has the gene (mecA), which codes penicillin-binding protein (PBP 2a), which has an alow affinity for β-lactam antibiotics (17). Many reports showed that the MRSA strains had been isolated from animals and humans causing infections, and MRSA has been able to transfer between dairy cows and the person milking them (18). The veterinarians, farm workers, and farm animals are exposed to the potential risk by direct contact with the infected cattle by the presence of MRSA in bovine milk (19). The previous studies declared that MRSA had been isolated from domestic animals such as veal calves (20), poultry (21) , horses (22), and camel (23).

This study aimed to detect S. aureus isolates using the traditional and molecular techniques on a specific gene nuc and investigate methicillin-resistant S. aureus by detecting the specific mecA gene for MRSA.

Materials and methods

Sampling

 Sixty-six samples were collected from the sub-clinical mastitis from November 2018 to February 2020 from different areas of the Nineveh Governorate. Milk samples were obtained after cleaning the teat, ant-dipping the teat, and discarding the first few streams of milk. The milk samples collected from subclinical bovine mastitis were 10 ml. All milk was placed into sterile glass vials. After dipping the teat detergent agents such as 70% alcohol were used to disinfect the teat from bacteria (24). The samples were kept on an icebox and transported to the Research Center and Laboratories, College of Veterinary Medicine, Mosul University. The milk samples were streaked onto Blood media (Lab M limited Topley house, Lancashire, United Kingdom) and Mannitol salt media 7.5% plates (118 g/L) (Lab M limited Topley house, Lancashire, United Kingdom). The culture plates were incubated at 37°C for 24 h.

Isolation and Identification of S. aureus

The typical S. aureus colonies were examined by gram staining and the traditional biochemical methods (catalase and coagulase test), and morphology (25).

DNA isolation

All the positive S. aureus isolates were grown on mannitol salt media for 24 h at 37°C. Genomics DNA of S. aureus was isolated based on manufacturer's protocol for Gram-positive bacteria, DNA of S. aureus isolated with the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). The amount of DNA extracted was weighted using Nanodrop (Biodrop, United Kingdom), and the DNA was stored at -20°C .

PCR Reaction

Based on the PCR assay, the specific-species nuc gene of S. aureus and the mecA gene of MRSA was detected. PCR interaction was implemented by the 200μl tube (Biozym, Oldenhorf, Germany) together the whole volume mishmash was 25 μL, consisting of 1 μL of each F and R primer (each ten pmol/μL), (Eurofins Genomics, Ebersberg, Germany) (Table 1). The molecular weight of the nuc gene is 166 bp (27), while the molecular weight of the mecA gene is 533 (28), 12.5 μL of 2×Go Taq Green Master Mix, and eight μL of double-distilled water (Promega). Eventually, a 2.5 μL DNA template of S. aureus or MRSAwas added to each 200 μl tube. The amplicons were determined using gel electrophoresis with a 100 bp ladder in 2% agarose gel (Peqlab, Erlangen, Germany).

Table 1: PCR programs and Primers in detecting of nuc and mecA of S. aureus and MRSA

PCR program^: I: 35 times (94°C - 30s, 55°C - 30s, 72°C - 30s), II: 35 times (94°C - 30s, 54°C - 30s, 72°C - 30s)

Results

The number of samples used in the current study collected from different regions in the Nineveh Governorate was 66. Regarding the guidelines of the National Mastitis Council (NMC) 2017 (29), S. aureus isolated from the milk samples was 23 (34.8%). All the positive S. aureus isolates had appeared the positive results with Gram, catalase, and coagulase tests. In addition, S. aureus was detected as round, golden-yellow clusters on mannitol salt media and hemolysis on the blood media. Furthermore, the study results demonstrated the nuc gene, which was detected in 23 (34.8%) of S. aureus (Figure 1). PCR assay was similar to the result of the phenotypic determination tests. In terms of the presence and absence, the mecA gene was seen in 12 (52.2%) of the 23 S. aureus isolates (Figure 2).

Figure 1: 2% of Agarose gel electrophoresis shows the product size of the nuc gene of the S. aureus isolates (166 bp).

Figure 2: 2% of Agarose gel electrophoresis shows an amplicon of the mecA gene product of the methicillin-resistant S. aureus isolates (533 bp).

Discussion

For several decades, S. aureus has been emergency pathogenic bacteria in human and animal importance fields. The current study used classical and molecular biological methods to isolate and identify MRSA in bovine milk. This study showed that the prevalence rate of S. aureus isolated from milk samples was 23 (34.8%). The result of the present study was nearest to the previous studies, which reported that the prevalence rate of S. aureus isolated from dairy mastitis milk was 35.9% and 36.3% in Egypt (30,31). While the result of this study was lower than other studies that reported the prevalence rate of S. aureus isolated from bovine mastitis milk was 74% in Egypt, 70% in Hungary, 43% in the USA (32-34), (33). In addition, the result of the present study was higher than other studies that reported the spread rate of S. aureus isolated from mastitis milk was 21.8% in Germany (35), and 5.6% in Korea (36). The prevalence rate of S. aureus is different based on the geographical distribution and the sanitary conditions in dairy farms, dairy plants. Many previous studies appeared that the S. aureus has isolated from skin of udder, bedding, workers' hands, insects, and dust that played an essential role in the transmission of S. aureus among cattle and contaminated milk (37). In addition, the isolation of S. aureus from the different organs of infected cows such as vagina, muzzle, skin wounds participates in the transmission of S. aureus between body sited in one hand and the environments on the other hand, as well as dairy herds, moreover, from cows to their calves by way of the ventilation or by feeding of milk having S. aureus (38).

Furthermore, the prevalence rate of MRSA isolated from bovine mastitis milk was 12 (52.2%). The result of the current study was higher than other studies that reported the spread rate of MRSA isolate was 28.2% in Egypt (39), 15% in Belgium (40), and 0.18% in Korea (41). MRSA is colonized and proliferated in the udder leading to cause the subclinical and clinical mastitis, which causes enormous economic loss (42). MRSA is transmitted from infected cattle to the calf (20). MRSA has isolated from humans, which contact domestic animals such as veterinarians, workers, owners (19). The molecular biology techniques were used to detect the mecA gene in S. aureus isolate, which was encoded for the synthesis to synthesize Penicillin-binding protein based on the (PCR) assay to detect and identify the target gene in the isolates (43).

Conclusion

1. aureus is regarded as a significant pathogenic microorganism to humans and ruminants. It can cause mastitis in mammals. MRSA possesses the mecA gene, causing problems to humans and animals during treatment because MRSA can resist the different types of beta-lactams antibiotics. PCR is an essential method for identifying the bacteria isolates and detecting the specific gene, and PCR is faster, simpler, and more accurate.

Acknowledgment               

The author is grateful to the University of Mosul /College of Veterinary Medicine for all the facilities to achieve this study.

Conflict of interest

There is no conflict of interest.