The quality and quantity of milk obtained from the cows is important for the dairy sector. Infection of mammary gland - mastitis is one of the biggest problems of dairy producers causes great losses every year in the livestock economy. The disease is usually local but may become systemic, although rarely, in immunocompromised animals (Tančin et al., 2006; Shari, Umer and Muhammad, 2009; Andrei et al., 2010; Vršková et al., 2015).

Mastitis is characterized by several physical and chemical alterations of the milk and corresponding pathological changes in the mammary tissue depending on the type of the disease. Intramammary infection (IMI) is caused by interaction of various factors associated with the animal, pathogens and the environment, so nature and duration of the disease varies accordingly (Taponen et al., 2006; Suriyasathaporn et al., 2006; Zajác et al., 2012).

More than 140 different microorganisms are recognized to cause mastitis. Infectious agents like bacteria, viruses, fungi and algae are mostly the primary causes of the disease.

Authors Vasiľ et al. (2009) and Tenhagen et al. (2006) indicate that up to 95% IMI is caused by bacterial pathogens. At routine bacteriological examination of milk from suspect cows, or secretion of the cow udder with clinical mastitis we can detect Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, CNS, Escherichia coli, Enterobacter spp., Klebsiella spp., Serratia spp., Pseudomonas spp. and others.

During a bacterial infection the number of somatic cells in milk increases, especially that of polymorphonuclear cells (PMN). The antibacterial activity of PMN cells is partially mediated through reactive oxygen species (ROS); an excess of these species correlating with the absence of optimal amounts of antioxidants can lead to oxidative stress. It was shown that the occurrence of oxidative stress in cattle may contribute to some periparturient disorders (retained fetal membranes, udder edema, mastitis) or metabolic diseases (Castillo et al., 2006; Sharma et al., 2011).

Oxidative stress in veterinary medicine and particularly in ruminant health is a relatively young field of research. Early identification of udder health problems is essential for dairy farmers and veterinarians to ensure not only the animal well-being but also the milk quality and dairying productivity. Economic aspects interfere with the routine application of bacteriologic examination of quarter milk samples. For this reason, alternative indicators of oxidative stress are used to identify trends in the development of the udder health in a dairy herd. Although these parameters belong detection of lipid peroxidation reactive aldehydes such as malondialdehyde (MDA) (Suriyasathaporn et al., 2012; Sharma et al., 2016).

MDA is one of the several low molecular weight end products formed during the radical induced decomposition of polyunsaturated fatty acid. MDA modifies the physical structures of cell membranes and is indirectly involved in the synthesis of protein, DNA, and RNA. In addition, it has mutagenic and carcinogenic properties (Turk et al., 2017; Kapusta et al., 2018).

Table 1 shows the health status udder of 223 lactating dairy cows. From (892 quarter milk samples were recorded in 161 positive samples (18.1%) with CMT score trace or 1 ˗ 4. Negative CMT score were recorded in 731 quarter milk samples (81.1%). A total of 127 infected quarter milk samples from 67 mastitis cows were the most commonly diagnosed form of SM 8.7% from all investigated quarters. At quarter levels, 16 (1.8%), 34 (3.8%) quarters were classified as latent and clinical mastitis, respectively. Numbers and percentages of isolates separated for their severity of mastitis are shown in Table 2. CNS gave the highest percentages (37%) representation on the etiology of mastitis in the monitored herd. SM were represented by up to 61.4% from all mastitis samples. The most common pathogens in CM were S. aureus, S. uberis and S. sanguinis. Other bacteria and S. intermedius had very few data and were excluded from analyses.

Averages of milk MDA for the selected groups and for the selected pathogens causing SM were shown in Figures 2(A) and (B), respectively. Averages, values of milk MDA in this study from health, subclinical and clinical group were 19.6, 28.8, and 41.3 nmol.mL^-1, respectively. From univariate analyses, results show that differences between selected groups and pathogens causing SM were related to MDA concentrations (p <0.001). Milk from SM and CM quarters had highest MDA concentrations and were statistically different (p <0.001) compared to healthy quarters (Figure 2(A)). MDA from milk samples with S. aureus, CNS, Str. sanguinis and Str. uberis was significantly higher compared to MDA from health milk samples (Figure 2(B)). Results from Post Hoc probabilities showed, that MDA from milk samples infected with Str. uberis were statistically higher (p <0.001) compared to other pathogens (Table 3).

Bacteria are the most common cause of mastitis and hence bacteriological culture is routinely used in the laboratory diagnosis. The bacterial pathogens isolated from 127 affected quarters were CNS (41.7%), followed by CPS (16.5%), Str. sanguinis (7.0%), Str. uberis (6.3%) and other bacteria with mixed infections (8.6%) (Table 2).

The present findings are in accordance with the findings of Kivaria and Noordhuizen (2007) isolated Staphylococcus spp. followed by Streptococcus spp., E. coli and Klebsiella.

Tenhagen et al. (2006) isolated Staphylococcus spp. followed by E. coli, streptococci and Pseudomonas.

In many countries, CNS have been acknowledged more and more frequently as a cause of intramammary infections in dairy cattle (Pyörälä and Taponen, 2009; Lange et al., 2015).

In the case of CNS, there has been not only an increasing prevalence of such infections but also an expanding list of species reported to be involved in the process. Results of studies from different countries and continents revealed that more than 20 CNS species have been isolated from milk samples of mastitic cows, the most common being S. chromogenes, S. haemolyticus, S. epidermidis, S. simulans and S. xylosus (Zadoks et al., 2011; Lange et al., 2015; Khazandia et al., 2018).

In our study the most common CNS species such as S. haemolyticus, S. chromogenes, S. warneri and S. xylosus have been isolated from infected quarter milk samples of cows.

According to Vasiľ et al. (2016) staphylococci and streptococci are the main etiological agents of ruminant IMI. Staphylococcus aureus with CNS are the most frequent isolates from subclinical and clinical cases of IMI.

Monday and Bohach (1999) present in their study that CNS are the most prevalent pathogens causing subclinical mastitis in dairy ruminants. Although less pathogenic than S. aureus, CNS can also produce persistent SM as well as producing thermostable enterotoxins. Nevertheless, despite the accepted role of these bacteria as major IMI causing pathogens in dairy ruminants, the pathogenicity of the different CNS species varies widely.

In our study, CNS, S. aureus and streptococci were most common isolated from SSM what is generally seen as an increase in the SCC in milk of the infected quarter with positive CMT score.

Increase in SCC during the inflammatory process in mammary glands indicates increased neutrophils in milk which result in oxidation reactions and increase MDA levels. In cows with mastitis, serum lipid peroxidation levels were increased, and the level of blood glutathione peroxidase was decreased compared to the levels in healthy cows (Atroshi et al., 1996).

According to our previous study Zigo et al. (2014) infected lactating cows are more sensitive to oxidative stress than cows without IMI. An imbalance between increased production of ROS and the availability of antioxidant defenses needed to reduce ROS accumulation during the infection and may expose cows to increased oxidative stress.

Lipid peroxidation is one of the important consequences of oxidative stress. MDA is generated as a consequence of lipid peroxidation and, as such, is assayed as a biomarker of oxidative stress. The significant higher changes in milk MDA concentration of cows with subclinical and clinical mastitis in the present study (Figure 2(A)) are in accordance with the study Suriyasathaporn et al. (2012) that showed a significant increase of milk MDA levels in cows with SM and CM.

According to our expectation, CM quarters had highest MDA concentrations. This might be caused by the higher levels of udder defence mechanism. With the perspective of early diagnosis have shown increased MDA levels in SM quarters. This study showed that MDA level was different among pathogens causing subclinical mastitis (Figure 2(B)). Characteristics of pathogens may be the reason for differences in oxidative environment in udders but MDA from milk samples wit Str. uberis were statistically higher compared to other pathogens (Table 3).

In the study, we confirmed that the current pathogens mammary gland includes CNS, S. aureus, S. sanguinis and S. uberis, which were most often isolated from SM and CM. The highest MDA level was observed from clinical cases of mastitis however, elevated levels of MDA were detectable from SM quarters. Bacterial isolates from subclinical quarter milk samples are different levels of MDA. In this study, we found that S. uberis was statistically higher compared to other pathogens.

Subclinical mastitis is difficult to detect because of a lack of clinical signs that can be easily identified by visual inspection and palpation of the udder. As can be seen from our study, one of the additional methods for detecting SM can be measurement of MDA level in raw cows’ milk.