Edyta Kaczorek

The effect of kynurenic acid on the synthesis of selected cytokines by murine splenocytes - in vitro and ex vivo studies.

Kynurenic acid (KYNA), a secondary product of the kynurenine pathway of tryptophan degradation, known mainly as an endogenous neuroprotectant, shows also immunotropic properties. Some quantities of KYNA are present in food and are effectively absorbed in the gastrointestinal tract. Since the spleen is an important target of dietary immunomodulators, the aim of the study was to determine the effect of exogenous KYNA on murine splenocytes. Splenocytes isolated from adult BALB/c mice were used in the study. Firstly, the effect of increasing KYNA concentrations (0-5 mM) on the viability, and proliferative and cytokine response (interleukin 1β [IL-1β], IL-6, IL-10, tumor necrosis factor α [TNF-α]) of murine splenocytes under in vitro conditions was determined. Then, proliferative and cytokine responses were determined in cells derived from animals receiving kynurenic acid in drinking water at concentrations of 2.5, 25, or 250 mg/l for 7-14 days. Cytokine levels were measured using commercial immunoassay (ELISA) kits, and cell viability and proliferation was determined with MTT reduction assay. Exogenous KYNA was characterised by a low level of cytotoxicity towards murine splenocytes, and was well tolerated by the animals receiving it in drinking water. As expected, it exhibited anti-inflammatory action towards the activated splenocytes, under both in vitro and ex vivo conditions. Surprisingly, however, KYNA itself influenced the activity of resting, non-stimulated cells, exerting an immunostimulant effect in vitro, and an immunosuppressive effect under ex vivo conditions. The obtained results indicate not only anti-inflammatory, but also more complex, immunomodulating properties of KYNA, which require more detailed investigation.

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 oral administration of kynurenic acid on the activity of the peripheral blood leukocytes in mice.

Kynurenic acid (KYNA), an endogenous tryptophan metabolite, is a selective ligand of the GPR35 receptor, expressed mainly on the immune cells. In inflammatory conditions, by affecting this receptor, KYNA inhibits the synthesis of pro-inflammatory cytokines and probably protects tissues from oxidative damage. However, we lack data regarding the effect of exogenous KYNA on the activity of immune cells in healthy individuals. The objective of this study has been to determine the influence of kynurenic acid administered to mice in different doses (2.5, 25 or 250 mg/l) and for different time periods (3, 7, 14, 28 days) in drinking water, on the activity of their peripheral blood leukocytes. The determinations comprised the proliferative activity of lymphocytes (MTT assay) and the phagocytic activity as well as the respiratory burst activity of granulocytes and monocytes (Phagotest, Phagoburst). It was only the lowest KYNA dose that influenced the mitogenic response of lymphocytes, namely by increasing the proliferation of T cells. The impact on the phagocytic activity was varied with KYNA dose and administration time. However, all the KYNA doses significantly lowered the activity of oxidative burst in phagocytes, which was probably associated with its antioxidant properties. In the light of the research results, kynurenic acid may find applications as an immuno-modulating agent able to correct an excessive or insufficient response of phagocytizing cells, protecting an organism from oxidative stress.

Biofilm production and other virulence factors in Streptococcus spp. isolated from clinical cases of bovine mastitis in Poland

BackgroundMastitis is a common disease in dairy cattle throughout the world and causes considerable economic losses each year. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among bacteria of the genus Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland.ResultsMost of the isolates analysed demonstrated an ability to produce biofilm (over 70%). Virulence genes were searched for in the three most common streptococci in our experiment: S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33%), cylE (78%), bca (37%), and cfb (100%). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes of the species were Ia (n = 8) and II (n = 8), in addition to some strains that were not classified in any of the groups (n = 6). Out of the eight selected genes for S. uberis (sua, pauA/skc, gapC, cfu, lbp, hasA, hasB, hasC), only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76% and 86% of the strains, respectively.ConclusionsThe experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.