Streptococci as the new dominant aetiological factors of mastitis in dairy cows in north-eastern Poland: analysis of the results obtained in 2013-2019.

The objective of our study was to evaluate prevalence of selected bacterial and fungal pathogens of mastitis in dairy cattle in north-eastern Poland. Our study was conducted from 2013 to 2019 in 1,665 clinically and sub-clinically infected quarter milk samples (2013, n = 368; 2014, n = 350; 2015, n = 290; 2016, n = 170; 2017, n = 173; 2018, n = 224; and 2019, n = 90). The isolation and identification of the pathogens were performed in keeping with generally accepted microbiological procedures. In 2013, mastitis was most commonly caused by Staphylococcus aureus (24%), Streptococcus spp. (22%), Streptococcus agalactiae (12%) and coagulase-negative staphylococci (11%). In 2014, the most common pathogens were Streptococcus spp. (25%), Staphylococcus aureus (18%) and coagulase-negative staphylococci (10%); in 2015, 2016, 2017, 2018 and 2019, Streptococcus spp. (from 39-49%) were the most frequent strains isolated from the quarter milk samples. Other pathogens were isolated occasionally (below 15% in all years). In conclusion, the role of environmental bacteria has been gradually increasing in the Warmia Province. The importance of infectious pathogens has been decreasing, indicating the efficacy of the applied preventive programmes and a need for the development of new programmes targeting environmental pathogens.

Dietary supplementation with Lactobacillus plantarum and ß-glucan affects immune parameters in the tench (Tinca tinca) fry.

The aim of the experiment was to examine the effect of a diet enriched with Lactobacillus plantarum and/or ß-glucan on the immune parameters in the juvenile tench (Tinca tinca). Fish were fed for 14 days different diets (phase 1 of the experiment), a dry commercial starter feed in the control group or the same feed supplemented with: 1% ß-1,3/1,6-glucan in group G, 108 cfu L. plantarum g-1 in group L, 1% ß-1,3/1,6-glucan + 108 cfu L. plantarum g-1 in group G+L. During consecutive 14 days all fish were fed the commercial feed alone (phase 2). The stimulating effects of the tested preparations was evaluated twice, at the end of each experimental phase. Dietary supplementation of ß-1,3/1,6-glucan considerably improved the humoral innate immune response (activity of lysozyme and total Ig) and the pinocytotic activity of phagocytes. Supplement of L. plantarum improved the ability of the head kidney phagocytes (RBA) to carry out oxygen burst in L and G+L groups. A similar effect was observed for the killing activity of phagocytes (PKA) from the head kidney after the stimulation of A. hydrophila, and the effect persisted for two weeks after the commercial feed regime was resumed. A significant increase in the proliferative activity of B lymphocytes originating from the head kidney was observed in groups L and G+L. The study has revealed that the addition of the tested G+L synbiotic to dry diet stimulates the innate immune response mechanisms in the juvenile tench.

The applicability of the Migratest® kit for evaluating the chemotactic activity of peripheral blood neutrophils in goats on the example of animals supplemented with ß-hydroxy-ß-methylbutyrate (HMB).

The objective of this study was to determine the applicability of the Migratest® kit for evaluating the chemotactic activity of peripheral blood neutrophils in goats. The experiment was performed on 14 goat kids aged 30±2 days, divided into two groups of 7 animals each: C - control group, and E - experimental group, supplemented with ß-hydroxy-ß-methylbutyrate (HMB), a typical immunostimulant which influences the phagocytic activity of peripheral neutrophils. The feed administered to experimental goat kids was supplemented with HMB at 40 mg/kg BW, whereas control goat kids were administered standard farm-made feed without supplementation. Blood was sampled from the jugular vein immediately before the experiment (day 0) and on experimental days 15, 30 and 60 to determine the chemotactic activity of peripheral blood neutrophils in goats. The results of the study indicate that the Migratest® kit can be used to evaluate the influence of immunomodulators on the chemotactic activity of peripheral blood neutrophils in goats. The results of the assay are most effectively presented by calculating the chemotactic index which accounts for the chemotaxis or migration of neutrophils in the presence or absence of a chemotactic factor, respectively, and the percentage of granulocytes that migrate towards fMLP. The results of both presentation methods appear to be identical.

CD4- and CD8-expressing cells found in the bovine and porcine anterior chamber of the eye.

The aim of the present study was to investigate whether the anterior chamber constitutes part of the normal migratory pathway of CD4+ and CD8+ lymphocytes in cattle and swine. The cells obtained from aqueous humor of cows and pigs were stained for CD4 and CD8 receptors, and subsequently analyzed with flow cytometry. The mean percentage of CD4+CD8-, CD4-CD8+ and CD4+CD8+ cells within the total lymphocyte population of the bovine anterior chamber was, respectively, 17.88, 12.64 and 27.26%. In turn, the mean values of these parameters in pigs were 1.77, 38.48 and 17.45, respectively. Among bovine and porcine CD4+CD8+ cells prevalent were those displaying CD4lowCD8low and CD4lowCD8high phenotypes, respectively. The results suggest that the anterior chamber in cattle and swine is an element in the normal migratory pathway of CD4+, CD8+ and CD4+CD8+ cells. Furthermore, the contribution of these subsets in the anterior chamber lymphocyte population can differ considerably between animal species.

Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.

Mastitis of dairy cattle is one of the most frequently diagnosed diseases worldwide. The main etiological agents of mastitis are bacteria of the genus Streptococcus spp., in which several antibiotic resistance mechanisms have been identified. However, detailed studies addressing this problem have not been conducted in northeastern Poland. Therefore, the aim of our study was to analyze, on phenotypic and genotypic levels, the antibiotic resistance pattern of Streptococcus spp. isolated from clinical cases of mastitis from dairy cattle in this region of Poland. The research was conducted using 135 strains of Streptococcus (Streptococcus uberis, n = 53; Streptococcus dysgalactiae, n = 41; Streptococcus agalactiae, n = 27; other streptococci, n = 14). The investigation of the antimicrobial susceptibility to 8 active substances applied in therapy in the analyzed region, as well as a selected bacteriocin (nisin), was performed using the minimum inhibitory concentration method. The presence of selected resistance genes (n = 14) was determined via PCR. We also investigated the correlation between the presence of resistance genes and the antimicrobial susceptibility of the examined strains in vitro. The highest observed resistance of Streptococcus spp. was toward gentamicin, kanamycin, and tetracycline, whereas the highest susceptibility occurred toward penicillin, enrofloxacin, and marbofloxacin. Additionally, the tested bacteriocin showed high efficacy. The presence of 13 analyzed resistance genes was observed in the examined strains [gene mef(A) was not detected]. In most strains, at least one resistance gene, mainly responsible for resistance to tetracyclines [tet(M), tet(K), tet(L)], was observed. However, a relationship between the presence of a given resistance gene and antimicrobial susceptibility on the phenotypic level was not always observed.

Effect of feed supplementation with kynurenic acid on the morphology of the liver, kidney and gills in rainbow trout (Oncorhynchus mykiss Walbaum, 1792), healthy and experimentally infected with Yersinia ruckeri.

Kynurenic acid (KYNA) is an endogenous substance produced on the kynurenine pathway which is primarily known for its neuroactive properties. Recently, it has been proven that KYNA is a selective ligand for G protein-coupled receptor (GPR 35), presented on immunocompetent cells such as T lymphocytes. This opens up new possibilities of its application as an immunostimulating substance in aquaculture. Thus far, no histopathological investigations in fish have been completed to evaluate influence of KYNA supplementation in feed. This study has been undertaken to determine the effect of feed supplementation with KYNA (2.5, 25, 250 mg kg-1 of feed) for 28 days on the liver, gills and kidney in healthy fish and experimentally infected with Yersinia ruckeri. In a control group were observed a fatty liver, which is natural for this fish species in the autumn and winter season. As the dose of the supplement was increased, the fat liver changed, it decreased or completely disappeared. Additionally, inflammatory changes occurred in all the analysed organs, and their intensification was dose dependent. In the fish experimentally infected, KYNA caused aggravation of the signs in the liver, kidneys and gills, and the effect was dose dependent. The results implicate that KYNA may be a stressor for fish.

Effect of oral administration of commercial gold nanocolloid on peripheral blood leukocytes in mice.

During the last few decades, owing to their unique properties, gold nanoparticles (AuNPs) have found numerous biomedical applications. Studies on rodents prove that AuNPs entering an organism easily reach the bloodstream and undergo wide tissue distribution. The presence of nanoparticles inside blood and bone marrow cells of exposed animals may implicate its influence on hematopoesis and the functions of peripheral blood leukocytes. The aim of this study was to determine the effect of oral administration of commercial gold nanocolloid, recommended by the producer as a dietary supplement, on the percentage of lymphocyte populations and proliferative response, as well as the activity of phagocytes in the peripheral blood of mice. The colloid was given to the animals in three different doses (0.25, 2.5, 25 ppm), for three different time periods (7, 14, 28 days). Mice given nanoparticles showed increased activity of phagocytes and some changes in the lymphocyte phenotypes. The elevated activity of granulocytes and monocytes, in terms of both phagocytic and respiratory burst activity, was transient and noticed only after a short time of administration, which may indicate some adaptability of blood phagocytes to prolonged presence of gold nanoparticles in the body. However, phenotypic modifications among lymphocytes in the group of animals given the middle dose of colloid (i.e. increased percentage of B and CD4+CD8+ DP T cells) did not occur until after the 28-day administration, which in turn seems indicative of some immune dysregulation due to the prolonged contact with nanogold.

The splenocyte proliferative response and cytokine secretion in mice after oral administration of commercial gold nanocolloid.

Owing to their unique physicochemical properties, gold nanoparticles find numerous biomedical applications. Experiments on rodents prove that the main target organs of gold nanoparticles entering an organism are the liver and spleen, whose reticuloendothelial system removes foreign particles from the bloodstream. Through interactions with resident tissue macrophages, nanoparticles can evoke a systemic immunological response. The aim of this study has been to determine the effect of oral administration of commercial gold nanocolloid, recommended by the producer inter alia as a dietary supplement, on the proliferative activity and cytokine secretion by murine splenocytes. The colloid was given to the animals in three different doses (0.25, 2.5, 25 ppm), for three different time periods (7, 14, 28 days). The influence of nanogold on splenocyte functions was time-dependent and the various doses were distinguished by distinct modes of action. The lowest dose had a pro-inflammatory or immunostimulating effect, enhancing the synthesis of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). The effect of the highest dose can be considered as a pro-inflammatory, or immunotoxic one, because the stimulated cytokine synthesis was accompanied by a drastic decline in the proliferative activity of lymphocytes. The medium dose, while inhibiting the synthesis of pro-inflammatory cytokines of macrophages, simultaneously stimulated the proliferation of lymphocytes. All the doses also modulated the synthesis of IL-2, which may implicate their effect on the immunoregulatory mechanisms of an organism. The effect of alimentary administration of gold nanocolloid on the immune system seems to be difficult to predict, hence a risk that this type of dietary supplements might have some adverse impact on the immunity cannot be excluded, especially after their chronic administration.

The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity.

Kynurenic acid (KYNA), an endogenous neuroprotectant formed along the kynurenine pathway of tryptophan degradation, is a selective ligand of the GPR35 receptor, which can be found on the surface of various populations of human immune cells. In infections and inflammations, KYNA produces an anti-inflammatory effect through this receptor, by depressing the synthesis of reactive oxygen species and pro-inflammatory cytokines. However, it is still unrecognized whether receptors for kynurenic acid are also localized on immune cells of poikilothermic animals, or whether KYNA is able to affect these cells. The objective of this study has been to determine the effect of different concentrations of kynurenic acid (12.5 microM to 10 mM) on the viability and mitogenic response of lymphocytes and on the activity of phagocytic cells isolated from blood and the spleen of rainbow trout. The results imply low toxicity of kynurenic acid towards fish immune cells, and the proliferative effect observed at the two lowest concentrations of KYNA (12.5 microM and 25 microM) seems indicative of endogenous kynurenic acid being capable of activating fish lymphocytes. Non-toxic, micromole concentrations of KYNA, however, had no influence on the mitogenic response of lymphocytes nor on the activity of phagocytes in rainbow trout under in vitro conditions. There is some likelihood that such an effect could be observed at lower, nanomole concentrations of KYNA.

Commercial metal-based nanocolloids--lack of virucidal activity against ECBO virus.

Metallic nanoparticles, mainly silver ones, have been widely used as antibacterial agents, and some studies shown they also exert direct antiviral activity against both enveloped and non-enveloped viruses. The objective of this study has been to evaluate the virucidal activity of commercial silver, gold, copper and platinum nanocolloids, recommended by the manufacturer as antimicrobials, against the ECBO virus, according to Polish Standard PN-EN 14675:2006. The highest experimentally observed decrease in the viral load was 0.875 log, which--when contrasted with the reduction in virus titre of at least 4 log expected from disinfectants--indicates that none of the analyzed nanocolloids had a disinfectant power towards the ECBO virus under the conditions defined by the standard.

Effect of 28-day oral administration of silver nanocolloid on the peripheral blood leukocytes in mice.

Silver nanoparticles, which have found a wide range of applications owing to their antimicrobial properties, are also recommended as dietary supplements in alternative medicine. Studies on rodents confirm that nanosilver is absorbed from the digestive tract into the bloodstream, which implies its possible interactions with leukocytes. The objective of the experiment discussed herein has been to determine the effect of 28-day oral administration of different doses (0.25, 2.5, 25 ppm) of commercial silver nanocolloid on hematological parameters, percentages of particular lymphocyte populations and activity of the peripheral blood leukocytes in mice. All the tested colloid doses decreased the counts of monocytes in the animals' blood and induced phenotypic modifications among lymphocytes: an increase in CD4+/CD8+ T cell distribution, a decrease in NK and NKT cell distribution (doses of 0.25 and 2.5 ppm) and an increased CD4+:CD8+ ratio (25 ppm). Silver nanocolloid also affected the activity of cells, depressing the proliferation of lymphocytes (0.25 ppm) and stimulating phagocytosis as well as the respiratory burst of granulocytes and monocytes (all doses). The results verify the influence of orally administered silver colloid on the peripheral blood leukocytes, at the same time implying the potential risk of developing an inappropriate immune response of an organism exposed to prolonged administration of this substance.

The effect of beta-hydroxy-beta-methylbutyrate (HMB) on selected parameters of humoral immunity in calves.

The objective of this study was to evaluate the effect of HMB on selected parameters of the humoral immunity in calves. The experiment was performed on 14 calves aged 30 +/- 2 days, divided into two equal groups of control (group K) and experimental (group H) animals. The feed administered to the experimental calves was supplemented with HMB at 40 mg/kg BW, whereas the control calves were administered standard farm-made feed without supplementation. Blood was sampled from the jugular vein immediately before the experiment (day 0) and on experimental days 15, 30 and 60 to determine the following immunological parameters: total protein levels, gammaglobulin levels, lysozyme activity and ceruloplasmin activity. An analysis of the results obtained revealed a significant increase (p < 0.05; p < 0.01; p < 0.001 respectively) in gammaglobulin levels and lysozyme activity throughout the entire experimental period, an increase (p < 0.05; p < 0.01 respectively) in ceruloplasmin activity on experimental days 15 and 30, but no changes in serum total protein levels of calves administered HMB as compared to those found in the control group.

The splenocyte proliferative response and cytokine secretion in mice after 28-day oral administration of silver nanocolloid.

An increasing number of applications of silver nanoparticles in industry, medicine and everyday life means that the risk of exposure of the human organism to their potential harmful influence is growing. This study has sought to assess the effect of 28-day alimentary administration of different concentrations (0.25, 2.5 and 25 ppm) of a commercial silver nanocolloid on the proliferative activity and synthesis of cytokines by mouse splenocytes. All of the analyzed doses of the colloid had a significant, albeit different, effect on the activity of splenocytes. At the lowest dose, a significant decrease in the proliferation of T cells and more intensive synthesis of pro-inflammatory cytokines, both by non-stimulated and LPS-stimulated cells, was observed. The intermediate dose, on the other hand, stimulated proliferation of B cells while producing a pro-inflammatory effect regarding the synthesis of cytokines. Finally, the highest dose decreased the synthesis of cytokines by non-stimulated cells, but after LPS stimulation, through the strong activation of the IL-10 synthesis, it raised the proliferation of B cells and decreased the synthesis of pro-inflammatory cytokines. The results suggest that silver nanoparticles administered orally have an easy access to the peripheral organs of the immune system, such as the spleen, but the effect of long-term exposure of this organ to the effect of silver nanocolloid depends on several factors, including the dose of nanoparticles, and seems as difficult to predict.

The in vitro effect of commercially available noble metal nanocolloids on the splenocyte proliferative response and cytokine production in mice.

Noble metal nanoparticles, currently among the most popular types of nanomaterials, are capable of penetrating through biological barriers once they enter a living organism. There, they can permeate into organs possessing the reticuloendothelial system, such as the spleen. The objective of this study was to determine the effect of commercial nanocolloids of noble metals (silver, gold and copper), recommended by the manufacturer as dietary supplements, on the in vitro viability, proliferative activity and production of cytokines (IL-1beta, IL-2, IL-6, IL-10 and TNF-alpha) by mouse splenocytes. All of the analyzed colloids had some effect on the activity of mouse splenocytes. Silver colloid was characterized by high toxicity - concentrations of 1.25 ppm and above substantially depressed the viability of cells as well as their proliferative activity and ability to synthesize cytokines. The other two colloids were far less toxic than nanosilver, although their non-toxic concentrations had a significant effect on the production of cytokines by mitogen activated splenocytes. The colloid of gold decreased the level of IL-2, and the colloid of copper caused an increase in IL-2, IL6 and IL-10. At the same time, copper colloid alone induced the synthesis of IL-1beta in mitogen unstimulated cells. The results indicate that colloids of noble metals are capable of affecting the activity of immunocompetent cells in important peripheral organs of the immune system.

The effect of beta-hydroxy-beta-methylbutyrate (HMB) on the proliferative response of blood lymphocytes and the phagocytic activity of blood monocytes and granulocytes in calves.

The objective of this study was to evaluate the effect of HMB on selected indicators of immunity in calves. The experiment was performed on 14 calves aged 30 +/- 2 days, divided into two equal groups of control (group I) and experimental (group II) animals. The feed administered to experimental group calves was supplemented with HMB at 40 mg/kg BW, whereas control calves were administered standard farm-made feed without supplementation. Blood was sampled from the jugular vein immediately before the experiment (day 0) and on experimental days 15, 30 and 60 to determine the following parameters of immunity: proliferative response of LPS- and ConA-stimulated lymphocytes (MTT), respiratory burst activity (RBA) and potential killing activity (PKA) of phagocytes. The results revealed a significant increase in RBA and MTT values in calves administered HMB in comparison with the control group throughout the experiment. In the group of animals receiving HMB, an increase in PKA values was noted only on day 30.

The in vitro effect of commercially available noble metal nanocolloids on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity.

The growing popularity of nanotechnology in the past decade has increased nanomaterial concentrations in the environment and the risk of their toxicity for aquatic organisms. Metal nanoparticles, which are easily absorbed and accumulated by fish, are probably able to interact directly with their immunocompetent cells. The objective of this study was to evaluate the in vitro effect of commercially available silver, gold and copper nanocolloids on the rainbow trout leukocyte and splenocyte activity. At high concentrations, all of the nanocolloids studied had adverse effects on the proliferative response of trout lymphocytes, and the most toxic of them, silver, decreased also the respiratory burst activity of splenocytes. Low concentrations of silver nanocolloid, however, had a stimulating effect on the lymhocyte proliferation.

The effect of silver nanoparticles on splenocyte activity and selected cytokine levels in the mouse serum at early stage of experimental endotoxemia.

The objective of this study was to determine the effect of a nonionic silver nanocolloid administered orally for 7 or 14 days at three concentration levels (25 ppm, 2.5 ppm, and 0.25 ppm) on the phagocytic activity and mitogenic response of splenocytes and selected cytokine serum levels (IL-1beta, IL-6, IL-10, IL-12 p70, TNF-alpha) in NMRI mice at the early stage of experimental endotoxemia induced with single 30 microg/mouse dose of bacterial LPS. Regardless of the period of administration, silver nanoparticles enhanced the production of proinflammatory cytokines and anti-inflammatory cytokine IL-10, and they inhibited IL-12 p70 levels in response to LPS challenge. The studied nanoparticles' effect on splenocyte activity was determined by the period of administration. After 7 days of use, silver nanoparticles enhanced the phagocytic activity, and doses of 2.5 ppm stimulated the mitogenic response of splenocytes. After 14 days of administration, silver nanoparticles lowered the phagocytic activity regardless of the dose applied. Although the results obtained are ambiguous, they suggest that silver nanoparticles administered via the alimentary tract are more likely to increase an inflammatory response of an organism than offer protection after LPS challenge.

The in vitro effect of bovine lactoferrin on the activity of organ leukocytes in rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis).

Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.

Immunomodulating effect of Inter Yeast S on the non-specific and specific cellular and humoral immunity in lambs.

The objective of this study was to determine the stimulating effect of the Inter Yeast S dietary supplement on selected parameters of specific and non-specific humoral and cellular immunity in lambs. The study involved 32 lambs aged 30 +/- 3 days, divided into two equal groups: II--control, and II--experimental. Experimental group animals were fed a C-J concentrate mixed with a prebiotic, the Inter Yeast S, commercially available, containing dried brewer's yeast Saccharomyces cerevisiae in the amount of 3 g/kg of the concentrate. At the beginning of the experiment (day 0) and on the 15th, 30th and 60th day of the study, blood was sampled from the jugular vein to determine selected parameters of biochemical, specific and non-specific humoral and cellular immunity in lambs (total protein levels, gamma globulin levels, lysozyme activity, ceruloplasmin activity, proliferative response of blood lymphocytes (MTT) after stimulation with LPS or ConA, the metabolic activity (RBA) and potential killing activity (PKA) of phagocytes). As regards humoral immunity parameters, significantly higher gamma globulin levels and higher lysozyme and ceruloplasmin activity were found in blood serum of experimental lambs administered the Inter Yeast S, compared with those determined in control lambs not fed the supplement. No statistically significant differences in serum total protein were found between the control and experimental groups. An analysis of cellular immunity indicators revealed significantly higher levels of RBA and PKA, and higher proliferative response of blood lymphocytes (MTT) after stimulation with LPS and ConA in the experimental group, compared with those observed in the control group.