Introduction. Pathological inflammatory processes in the mammary gland, especially those of a latent nature, are one of the factors that negatively affect the growth of cow milk production and the sanitary quality of milk in farms with different forms of ownership. Subclinical mastitis is one of the most common diseases, accounting for 20 to 80% of all inflammatory processes of the breast, according to various authors. Significant economic losses caused to dairy cattle as a result of this pathology are primarily due to a decrease in milk production of cows, culling of animals (30-35%) and treatment costs [1–3].
In addition to economic losses, the threat is posed by contamination of milk with pathogenic microorganisms, changes in the chemical composition, physical and biochemical properties of milk, resulting in a loss of nutritional value, which affects its quality and biological safety. Feeding colostrum from cows with mastitis to newborns can lead to gastrointestinal disorders and even death [4, 5].
In the treatment of sick cows, in most cases, preference is given to the use of antibiotics (intracisternal) and sulfonamide drugs. The most negative consequence of the use of antibiotics in the treatment of cows with mastitis is the presence of their residues in the collected milk, which worsens its technological properties and harms human health [6].
In recent years, scientific research on the use of liposomal preparations, which do not contain antibiotics, help prevent disease recurrence and maximize milk production, has expanded significantly. Liposomes are spherical lipid vesicles, which usually contain various phospholipids, specific glycopeptides, and cholesterol, that is a steroid component of almost all cell membranes [7].
The purpose of the research was to study the effect of a liposomal preparation based on ethylthiosulfanilate on the natural immunity of cows with subclinical mastitis.
Materials and methods. The study was conducted on cows of 2-3 lactation, which were divided into two groups according to the principle of analogues: control and experimental, 5 animals each. Subclinical mastitis in cows was diagnosed using Kenotest CID LINES N. V. immediately after milking. The number of somatic cells in the milk samples was determined using the LACTOSCAN SCC COMPACT somatic cell analyzer. The material for the laboratory tests was the blood of clinically healthy animals and whole milk, in which the number of somatic cells did not exceed 200 thousand/cm³ (control group). The experimental group was formed from animals with subclinical mastitis (SM), the number of somatic cells in the milk of these animals was in the range of 500 thousand up to 1 million per 1 cm³. The cows of the experimental group were injected intracisternal into the affected quarters of the udder three times with an interval of 24 hours – the first day 10 cm3, the next two days – 5 cm3. Half of the treatment dose was prophylactically injected into the healthy quarters of the breast. A liposomal preparation is an antibacterial drug that does not contain any antibacterial agents: thiosulfonic acid ethyl ester, vitamin A, vitamin D3, vitamin E, lecithin, tween, water for injection. The drug is active against gram-positive bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. The anti-inflammatory effect is due to the presence of flavonoids in the drug. It has the ability to heal the wound surface and stimulates tissue regeneration.
Blood from cows was collected from the jugular vein before morning feeding in two tubes on day 1 (before drug direction), day 3 and day 9 after drug administration. In the heparin-stabilized blood, phagocytosis parameters were determined – neutrophil phagocytic activity (PA). For a more complete characterization of phagocytosis, we determined the phagocytic absorption capacity (PI) by dividing the number of phagocytized microbial bodies by the PA. It was determined in the blood serum: bactericidal activity, lysozyme activity by photo nephelometric method, circulating immune complexes [8].
The obtained digital data were statistically processed using the Microsoft Excel software package for personal computers, using generally accepted methods of variation statistics with the determination of mean values (M), their squared error (m) and the reliability of differences by Student's t-test.
Results and discussion. The body's resistance is provided by both specific and nonspecific defense factors, and the body's natural immunity is determined by a complex of cellular and humoral factors. Phagocytosis is the main mechanism of natural resistance, as well as an essential link in the induction and formation of a specific immune response. One of the main components of the body's natural cellular defense is the phagocytic activity of neutrophilic granulocytes. Studies have shown that the disease of cows with a subclinical form of mastitis leads to changes in the phagocytosis of neutrophils in the blood of animals (Table 1).
Table 1. Dynamics of blood neutrophil phagocytosis in cows with subclinical mastitis (М±m; n=5)
Note. In the table: ˚ - P<0.05 - significance in animals of this group compared to the values before drug administration (day 1 of the experiment); * - P<0.05 - the difference is significant compared to the control group.
In particular, a higher 47±1.78% vs. 41±1.68%, P<0.05 PA of neutrophilic granulocytes in the blood of diseased cows was found compared to the control group against the background of a decrease in phagocytic number (FN) 4.15±0.28 units vs. 4.53±0.42 units and phagocytosis intensity in cows with latent mammary inflammation, which is 16.5% lower than in clinically healthy animals.
The administration of the experimental drug caused a decrease in the PA of granulocytes in the blood of the experimental group. Thus, already on the third day of treatment, the activity of phagocytic cells gradually decreased and this trend continued until the end of the treatment, indicating a complete extinction of the inflammatory response. Along with this, an increase in the phagocytic index and phagocytic count was found, which indicates a normalizing effect of the drug on the cellular link of the body's natural resistance. Studies of the state of nonspecific resistance of the body of cows with latent mastitis showed a violation of the components of the humoral link of immunity (Table 2).
Table 2. Humoral factors of blood protection in cows with subclinical mastitis (Мm; n=5)
Note. In the table: ˚ - P<0.05, ˚˚ - P<0.01 - significance in animals of this group compared to the values before drug administration (day 1 of the experiment); * - P<0.05, ** - P<0.01 - difference is significant compared to the control group.
Thus, the subclinical inflammatory process in the mammary gland was manifested by a decrease in the level of bactericidal activity (34.9±1.77% vs. 43.3±1.2%, P<0.01) and serum lysozyme activity (21.7±1.29% vs. 28.7±1.38%, P<0.01)). The conducted studies revealed an extraordinary antigenic load and accumulation of inflammatory metabolites in the body of sick cows, which was manifested by a significant increase in the number of circulating immune complexes (86.5±2.62% vs. 73.5±2.5%, P<0.01).
After administration of the liposomal preparation on day 9, an increase in the bactericidal activity of blood serum in animals of the experimental group was noted (42.4±1.12% vs. 34.9±1.77%, P<0.01), and a tendency to increase the lysozyme activity of cow serum was also noted (27.3±1.18% vs. 21.7±1.29%, P<0.05). Due to the restoration of metabolic processes, the concentration of circulating immune complexes in the blood serum of animals of the experimental group on day 9 of the experiment significantly decreased 74.8±1.1 vs. 86.5±2.62 mol/L, P<0.01).
Thus, the use of a liposomal preparation for the treatment of cows with subclinical mastitis helps to restore metabolic processes and normalizes the indicators of cellular and humoral defense factors of the animal body.
Conclusions. The disease of cows with subclinical mastitis leads to a statistically significant increase in phagocytic activity and circulating immune complexes and a decrease in bactericidal and lysozyme activity of blood serum.
Intracisternal direction of a liposomal preparation based on ethylthiosulfanilate to sick cows resulted in a decrease in the phagocytic activity of granulocytes in the blood. Along with this, an increase in the phagocytic index and phagocytic count was found. An increase in humoral indices of bacterial activity and lysozyme activity in the serum of diseased animals and a decrease in circulating immune complexes were also noted.
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