The CMT results of the study groups are presented in Table 1. All the quarters of the 10 animals in Group 1 were CMT and bacteriology negative.
Table 1: Determination of cows with sub-clinical mastitis.
In Groups 2-5, the number of positives CMT were according to scheduling 1-4 quarters respectively. In milk samples taken from CMT positive quarters, aerobic bacteria were isolated in 8 (80%) in Group 2, in 14 (70%) in Group 3, in 23 (76.6%) in Group 4, and in 32 (80%) in Group 5. When evaluated as a total, aerobic bacteria isolation was achieved in 77 (77%) of 100 CMT positive quarters of 40 lactating cows.
Bacteria isolated from cultivated milk
The bacteria isolated from the milk sample cultures according to the groups are presented in Table 2. The micro-organisms isolated from 77 milk samples were as follows: Staphylococcus aureus
in 35 (45.4%), Streptococcus agalactiae
in 15 (19.4%), Streptococcus dysgalactiae
in 10 (12.9%), Micrococcus spp
. in 4 (5.1%), Coagulase Negative Staphylococcus
(CNS) in 3 (3.8%), Corynebacterium bovis
in 3 (3.8%), Escherichia coli
in 3 (3.8%), Bacillus spp
. in 1 (1.2%), Arcanobacterium pyogenes
in 1 (1.2%), Pasteurella multocida
in 1 (1.2%) and Citrobacter diversus
in 1 (1.2%).
Table 2: Bacteria species isolated with MALDI-TOF MS (Matrix-mediated laser desorption ionization-flight time mass spectrometry database v2.0, bioMerieux, France).
In a previous study, the rates of micro-organisms isolated and identified from 125 CMT positive quarter milk samples were as follows: 47.3% Staph. aureus
, 16.3% C. pyogenes
, 8.2% E. coli
, 6.5% Candida albicans
, 6% S. agalactiae
and 15.7% other micro-organisms (Ates et al., 1991).
In other studies, Staph. aureus
has been reported to be the main pathogen most frequently isolated in sub-clinically infected quarters in Belgium and other regions and countries (Sampimon et al., 2005; Pitkale et al., 2004; Wilson et al., 1997).
Similar to the findings of previous research, the present study showed that Staph. aureus
appears to be the most commonly isolated bacteria in milk with sub-clinical mastitis. When other factors were considered, although the rates varied, the bacteria isolated were similar to those in other studies. Changes in the prevalence of Staph. aureus in the herd can be explained by differences between herds regarding post-milking teat dipping and dry-term udder treatment (Hogan et al., 1987).
Studies have reported that the prevalence of Str agalactiae varies between 8.5% (Wilson et al., 1997),
0.1% (Pitkale et al., 2004)
and 1-2% (Andersen et al., 2003).
In the present study, this rate was found to be 19.4%. This situation, which is contrary to the literature, was thought to be due to the fact that the mammary health control program was not fully implemented on the farm where the study was conducted. In addition, coliform bacteria, which are generally included in short-term acute clinical mastitis cases, have been reported to have a low prevalence in parallel with the current study (Hoblet et al., 1991).
Some blood parameters
The blood biochemistry values are presented in Table 3. Glucose and total cholesterol were significantly higher in Group 5 than in Groups 1, 2, 3 and 4 (P
Table 3: Blood biochemical parameters in the groups.
There was no significant difference between Groups 1, 2, 3 and 4 (P
>0.05). Total bilirubin was significantly lower in Group 1 compared to Groups 2, 3, 4 and 5 (P
<0.001). It was significantly lower in Group 2 than in Groups 4 and 5 (P<0.001). It was significantly lower in Group 3 than in Group 5 (P
<0.001). There was no significant difference between Groups 2 and 3, Groups 3 and 4 and Groups 4 or 5 (P
In this study, the relationship was evaluated between blood-biochemistry values such as glucose, T-cholesterol, T-bilirubin, AST and ALT, measured to assess liver function. Previous studies have reported that activities of AST and ALT enzymes increase in accordance with the elevation in CMT score (Qayyum et al., 2018).
In another study, milk and blood AST and ALT activities were higher in camels with sub-clinical mastitis compared to healthy animals (Ali et al., 2016).
In the current study, the increase in the values measured from the cows with sub-clinical mastitis was seen to be consistent with the data found in literature. When the effect of the number of infected quarters on the blood-biochemistry values is examined, glucose and T-cholesterol levels were determined to be lowest in Group 1 and highest in Group 5. While there was no statistically significant difference in these measurements between Groups 1, 2, 3, and 4, the difference between these groups and Group 5 was statistically significant, suggesting that the liver metabolic load may have increased due to the number of affected quarters. Similarly, T-bilirubin, AST and ALT were measured at the lowest activity in Group 1 and at the highest level in Group 5. The difference in the total bilirubin levels between Group 1 and the other groups (with sub-clinical mastitis) was found to be increasingly significant. The AST and ALT activities were significantly different in all the groups as the number of infected quarters increased. With the increase in the number of infected quarters, so the activities of liver enzyme parameters increased significantly (Ali et al., 2016).
This increase was thought to be related to microcirculation permeability as a result of tissue damage caused by free radicals in the quarter. It was thought that evaluations of sub-clinical mastitis should be made not only on an animal basis but also in terms of infected quarters.
Findings related to somatic cell count (SCC)
Ten cows in each group; from all CMT negative quarters in Group 1, 10 CMT positive in Group 2, 20 CMT positive in Group 3, 30 CMT positive in Group 4 and 40 CMT positive quarters in Group 5 SCC levels were established by taking the average of milk samples. When the SCC and CMT results were compared, the average SCC in the quarters in Group 1, which was CMT negative, was 155.05 cells/ml, 792.08 cells/ml in Group 2, 754.55 cells/ml in Group 3, 605.84 cells/ml in Group 4, and 695.33 cells/ml in Group 5 quarters. The difference in mean SCC values between Group 1 and the other groups was statistically significant (P
<0.001), and no significant difference was observed between the sub-clinical mastitis groups (P
>0.001) (Fig 1).
Total oxidant capacity (TOC), total antioxidant capacity (TAC) and oxidative stress index (OSI) measurements
Fig 1: The number of somatic cell count (SCC) in the groups. ***: P<0.001. The values in the column chart represent the mean ± SEM.
Total Oxidative Capacity (TOC) and oxidative stress index value (OSI) levels were significantly lower in Group 1 than in Groups 2, 3, 4 and 5 (P
<0.001), while Total Antioxidant Capacity (TAC) levels were significantly higher (P
<0.001), (Fig 2, Fig 3, Fig 4).
Fig 2: Total oxidant capacity (TOC) in the groups. ***: P<0.001. The values in the column chart represent the mean ± SEM.
Fig 3: Total antioxidant capacity (TAC) in the groups. ***: P<0.001. The values in the column chart represent the mean ± SEM.
Fig 4: Oxidative stress index (OSI) value in the groups (arbitrary unit). ***: P<0.001. The values in the column chart represent the mean ± SEM.
Studies on oxidative stress are among current topics and many scientific studies have been conducted in this area (Abuelo et al., 2013).
Oxidative stress is defined as an imbalance in favor of oxidants between oxidant and antioxidant substances (Puppel et al., 2015).
When free radicals and antioxidant substances encounter each other in the body, antioxidants prevent damage by inhibiting target molecules or by delaying oxidation thereby protecting the organism against tissue damage (Puppel et al., 2015).
Antioxidants have been found to have an antilipolytic effect, but free radicals disrupt the insulin mechanism, thereby stimulating lipolysis. In the case of oxidative stress, lipolysis is constantly active, and liver metabolic load increases in this case (Abuelo et al., 2016).
Tissue damage caused by intramammary inflammation results in the generation of reactive oxygen species (ROS), increasing the incidence of oxidative stress in mammary tissue, which can lead to increased permeability of microcirculatory arteries due to free radical injury. In a previous study investigating the oxidant/antioxidant status in cows with sub-clinical mastitis, it was emphasized that MDA and NO levels were higher than that of healthy control groups, and TAC and GSH levels were lower, indicating the contribution of udder-related oxidative stress and potential oxidative damage (Saleh et al., 2022; Sadek et al., 2017).
Similarly, it has been reported that MDA and H2
(Hydrogen peroxide) levels are increased and TAC levels are decreased in blood and milk serum of cows with sub-clinical mastitis compared to the control group (Nedić et al., 2019).
Moreover, TAC and MDA concentrations have been reported to increase significantly over time in untreated cows with sub-clinical mastitis (Tabatabaee et al., 2021).
Higher SCCs have also been strongly correlated with higher MDA levels (Yakan et al., 2021).
Total antioxidant capacity can be used to provide a simple understanding of the antioxidant status in the body (Farghali et al., 2021; Abdel-Saeed and Salem, 2019)
. The importance of TAC and TOC levels in monitoring oxidative stress-related diseases such as mastitis and the transition period in dairy cows has been emphasized (Amiri et al., 2020; Kurt et al., 2019; Turk et al., 2017; Andrei et al., 2016; Aydilek et al., 2014; Atakisi et al., 2010; Mandebvu et al., 2003).
In previous studies, milk with sub-clinical mastitis has been compared with cow and goat milk samples without sub-clinical mastitis and milk with mastitis has been reported to have higher TOC levels and lower TAC levels (Silanikove et al., 2014; Atakisi et al., 2010).
In another study, the TAC level was high in low-SCC milk and low in high-SCC milk in the samples taken according to the density of the somatic cells (Nedić et al., 2019; Andrei et al., 2016).
In our study, the serum TOC-TAC level was measured according to the number of infected quarters and the increase in TAC level and decrease in TOC level in animals with sub-clinical mastitis were consistent with the literature data (Silanikove et al., 2014; Atakisi et al., 2010).
The use of antioxidants increases as a result of the effect of free radicals that occur due to inflammation, which occurs in mastitis cases, and as a result, the levels of antioxidants decrease. In mammary gland inflammation, more oxygen is used depending on the activity of phagocytic cells that have migrated of phagocytic cells to the site of inflammation. The oxidative stress parameter measurements were determined to have a strong relationship with the number of infected quarters.