Proteome and Peptidome Changes and Zn Concentration in Chicken after In Ovo Stimulation with a Multi-Strain Probiotic and Zn-Gly Chelate: Preliminary Research.

The aim of the study was to determine differences in the proteome and peptidome and zinc concentrations in the serum and tissues of chickens supplemented with a multi-strain probiotic and/or zinc glycine chelate in ovo. A total of 1400 fertilized broiler eggs (Ross × Ross 708) were divided into four groups: a control and experimental groups injected with a multi-strain probiotic, with zinc glycine chelate, and with the multi-strain probiotic and zinc glycine chelate. The proteome and peptidome were analyzed using SDS-PAGE and MALDI-TOF MS, and the zinc concentration was determined by flame atomic absorption spectrometry. We showed that in ovo supplementation with zinc glycine chelate increased the Zn concentration in the serum and yolk sac at 12 h post-hatch. The results of SDS-PAGE and western blot confirmed the presence of Cu/Zn SOD in the liver and in the small and large intestines at 12 h and at 7 days after hatching in all groups. Analysis of the MALDI-TOF MS spectra of chicken tissues showed in all experimental groups the expression of proteins and peptides that regulate immune response, metabolic processes, growth, development, and reproduction.

The effect of psychoactive bacteria, Bifidobacterium longum Rosell®-175 and Lactobacillus rhamnosus JB-1, on brain proteome profiles in mice.

RATIONALE: The gut microbiota may play an important role in the development and functioning of the mammalian central nervous system. The assumption of the experiment was to prove that the use of probiotic bacterial strains in the diet of mice modifies the expression of brain proteins involved in metabolic and immunological processes. OBJECTIVES AND RESULTS: Albino Swiss mice were administered with Bifidobacterium longum Rosell®-175 or Lactobacillus rhamnosus JB-1 every 24 h for 28 days. Protein maps were prepared from hippocampal homogenates of euthanized mice. Selected proteins that were statistically significant were purified and concentrated and identified using MALDI-TOF mass spectrometry. Among the analysed samples, 13 proteins were identified. The mean volumes of calcyon, secreted frizzled-associated protein 3, and catalase in the hippocampus of mice from both experimental groups were statistically significantly higher than in the control group. In mice supplemented with Lactobacillus rhamnosus JB-1, a lower mean volume of fragrance binding protein 2, shadow of prion protein, and glycine receptor α4 subunit was observed compared to the control. CONCLUSION: The psychobiotics Bifidobacterium longum Rosell®-175 and Lactobacillus rhamnosus JB-1enhances expression of proteins involved in the activation and maturation of nerve cells, as well as myelination and homeostatic regulation of neurogenesis in mice. The tested psychobiotics cause a decrease in the expression of proteins associated with CNS development and in synaptic transmission, thereby reducing the capacity for communication between nerve cells. The results of the study indicate that psychobiotic bacteria can be used in auxiliary treatment of neurological disorders.

Effect of In Ovo Administration of a Multi-Strain Probiotic and Zinc Glycine Chelate on Antioxidant Capacity and Selected Immune Parameters in Newly Hatched Chicks.

The aim of this study was to determine the effect of in ovo co-supplementation of chicken embryos with a multi-strain probiotic containing effective microorganisms and zinc glycine chelate on total antioxidant capacity; concentrations of sulfhydryl groups, bityrosine bridges, formylkynurenines, hydroperoxides, proteins, corticosterone, pro- and anti-inflammatory cytokines and heat shock proteins; and the activity of catalase and superoxide dismutase in the serum, yolk sac and tissues of broiler chickens at 12 h and at 7 days after hatching. The results indicate high SOD activity in the small and large intestines of chicks at 12 h post-hatch in the groups receiving the multi-strain probiotic and in the small intestine and yolk sac of birds receiving the multi-strain probiotic and Zn-Gly chelate. High concentrations of TNF-α and IFN-γ in the yolk sac and serum after in ovo administration of Zn-Gly chelate were observed 12 h after hatching. The use of a probiotic and a probiotic with Zn-Gly chelate increased the total antioxidant capacity in the tissues of chickens. It can be concluded that in ovo administration of a multi-strain probiotic and Zn-Gly chelate can maintain the oxidant/antioxidant balance in chickens and increase the defense capacity against oxidative stress.

Effect of combined in ovo administration of zinc glycine chelate (Zn-Gly) and a multistrain probiotic on the modulation of cellular and humoral immune responses in broiler chickens.

The aim of the study was to determine the effect of in ovo administration of zinc glycine chelate (Zn-Gly), and a multistrain probiotic on the hatchability and selected parameters of the cellular and humoral immune response of chickens. The study was conducted on 1,400 fertilized eggs from commercial broiler breeders (Ross x Ross 708). Material for the study consisted of peripheral blood and spleens of chicks taken 12 h and 7 d after hatching. The results showed that both combined and single in ovo administration of the multistrain probiotic and zinc glycine chelate significantly reduced hatchability of chicks. The flow cytometry study showed that the highest percentage of CD4+ T cells, CD4+CD25+, and high expression of KUL01 in the serum were obtained in the group supplemented with probiotic and Zn-Gly both 12 h and 7 d after hatching. In birds supplemented with probiotic and zinc chelate, a high percentage of TCRγδ+ cells was found in serum and spleen 12 h after hatching and in serum after 7 d. The percentage of Bu-1A+ lymphocytes in serum and spleen 12 h and 7 d after hatching was the highest in the group supplemented with probiotic and Zn-Gly. The highest expression of CD79A was observed in the group supplemented only with zinc chelate. There were no significant differences in the percentage of CD4+ cells in the spleens of birds in the groups receiving the multistrain probiotic at 12 h after hatching, and after 7 d, the percentage of CD4+ T cells was lower in the experimental groups than in the control group. The percentage of CD8+ cells in the serum of birds after hatching was lower in the group supplemented with multistrain probiotic and Zn-Gly than in the control group, but reached the highest value on d 7 after hatching. The obtained results confirm the strong effect of the combined administration of a multistrain probiotic and Zn-Gly chelate on lymphocyte proliferation and stimulation of cellular immune mechanisms in birds.

The Effect of Feed Supplementation with EM Bokashi® Multimicrobial Probiotic Preparation on Selected Parameters of Sow Colostrum and Milk as Indicators of the Specific and Nonspecific Immune Response.

The aim of the study was to determine the effect of EM Bokashi® on selected parameters of the specific and nonspecific immune response of sows by in colostrum and milk samples. The percentage of cells with expression of CD19+, CD5+CD19+, CD21+, SWC3a (macrophage/monocyte), and CD11b+ molecules on the monocytes and granulocytes as well as the concentrations of lysozyme and acute phase proteins - serum amyloid-A (SAA) and haptoglobin (Hp) were evaluated. The study was carried out on a commercial pig farm, including 150 sows (Polish Large White × Polish Landrace) at the age of 2-4 years. Sixty female sows were divided into two groups: I - control and II - experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® in the amount of 10 kg/tonne of feed was added to the basal feed from mating to weaning. The material for the study consisted of colostrum and milk. The samples were collected from all sows at 0, 24, 48, 72, 96, 120, 144, and 168 h after parturition. The study showed that exposure of the pregnant sow to the probiotic microbes contained in EM Bokashi® significantly affects the immunological quality of the colostrum and milk and caused an increase in the percentage of the subpopulations of B cells with CD19+, CD21+, and CD5+CD19+ expression in the colostrum and milk, which demonstrates an increase in the protective potential of colostrum and indicates stimulation of humoral immune mechanisms that protect the sow and the piglets against infections.

Assessment of Antimicrobial Susceptibility of Virulent Strains of Rhodococcus equi Isolated From Foals and Soil of Horse Breeding Farms With and Without Endemic Infections.

Rhodococcus equi is an opportunistic, intracellular saprophyte that causes severe pyogranulomatous pneumonia in foals. The bacterium displays in vitro susceptibility to many antibiotics. The highest efficacy against R. equi in vitro and in vivo is achieved by using a combination of rifampicin and macrolide antibiotics. Recent years have seen an upward trend in the minimum inhibitory concentration (MIC) of rifampicin and erythromycin, suggesting increasing resistance of R. equi to these antibiotics. The aim of the study was to determine the antimicrobial activity of 24 selected antibiotics against R. equi strains isolated from dead foals and from the environment of horse breeding farms with and without endemic R. equi infections. Minimum inhibitory concentration gradient strips were used to determine the lowest concentration of the antibiotic that inhibited the growth of R. equi. Based on normal MIC distribution, an epidemiologic cutoff values (ECOFF) were assessed for particular antibiotics and R. equi strains. The results were compared with ECOFFs for S. aureus, according to the European Committee on Antimicrobial Susceptibility Testing data. The data indicate that the lowest MIC values were obtained for clarithromycin, rifampicin, imipenem, and vancomycin. The majority of R. equi strains can be classified as wild type in relation to the majority of antibiotics. A small percentage of strains presented non-WT (NWT) with the exception of SXT, for which 35% of strains were classified as NWT. The lack of interpretative criteria for R. equi creates a real problem in the assessment of antibiotic sensitivity both for clinical and scientific purposes.

The effect of feed supplementation with Transcarpathian zeolite (clinoptilolite) on the concentrations of acute phase proteins and cytokines in the serum and hepatic tissue of chickens.

The aim of the this study was to determine the effect of different levels of Transcarpathian zeolite (clinoptilolite) on selected indicators of the immune response in chickens by assessing the concentrations of the acute phase proteins haptoglobin, C-reactive protein (CRP), serum amyloid A, transferrin, and alpha-1-acid glycoprotein and the cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-2, and IL-10 in the serum and hepatic tissues of chickens. The study was conducted on 450 one-day-old male Ross 308 chickens. The total rearing period was 42 D. The samples of blood and liver were collected before the start of the study (day 0) and on day 42, after rearing was completed. ELISA kits specific for chicken CRP, haptoglobin, alpha-1-acid glycoprotein, serum amyloid A, transferrin, TNF-α, IFN-γ, IL-2, and IL-10 were used to determine the levels of acute phase proteins and cytokines in the serum and liver homogenates. The results of immunological tests suggest that for long-term maintenance of homeostasis in chickens, the addition of 2% zeolite as a feed additive is most beneficial. The results indicate that 3% clinoptilolite induce production of Th1 pro-inflammatory cytokines, increasing the synthesis of IL-2, IFN-γ, and TNF-α. The high concentration of IL-10 after the use of zeolite in conjunction with the high concentration of IL-2, TNF-α, and IFN-γ indicates a reduction in the intensity of inflammatory processes, the enhancement of the humoral immune response, and the simultaneous inhibition of the production of Th1-type cytokines. The increase of CRP concentration in conjunction with high concentrations of pro- and anti-inflammatory cytokines in the birds from the group receiving 3% clinoptilolite demonstrates indicates that it can influence the development of local inflammatory processes and enhance immune regulation in birds. Our research has shown that clinoptilolite influences on an increase in birds' resistance to infection, as confirmed by clinical observations and anatomopathological examination and by the increase in the synthesis of acute phase proteins with immunoregulatory properties.

Effect of Multi-Microbial Probiotic Formulation Bokashi on Pro- and Anti-Inflammatory Cytokines Profile in the Serum, Colostrum and Milk of Sows, and in a Culture of Polymorphonuclear Cells Isolated from Colostrum.

The use of probiotics in sows during pregnancy and lactation and their impact on the quality of colostrum and milk, as well as the health conditions of their offspring during the rearing period, are currently gaining the attention of researchers. The aim of the study was to determine the effect of Bokashi formulation on the concentrations of pro- and anti-inflammatory cytokines in the serum of sows during pregnancy, in their colostrum and milk, and in a culture of Con-A-stimulated polymorphonuclear cells (PMNs) isolated from the colostrum. The study was conducted on 60 sows aged 2-4 years. EM Bokashi were added to the sows' feed. The material for the study consisted of peripheral blood, colostrum, and milk. Blood samples were collected from the sows on days 60 and 114 of gestation. Colostrum and milk samples were collected from all sows at 0, 24, 48, 72, 96, 120, 144, and 168 h after parturition. The results indicate that the use of Bokashi as feed additives resulted in increased concentrations of pro-inflammatory cytokines TNF-α and IL-6, which increase the protective capacity of the colostrum by stimulating cellular immune mechanisms protecting the sow and neonates against infection. At the same time, the increased concentrations of cytokines IL-4, IL-10, TGF-ß, and of immunoglobulins in the colostrum and milk from sows in the experimental group demonstrate the immunoregulatory effect of Bokashi on Th2 cells and may lead to increased expression of regulatory T cells and polarization of the immune response from Th1 to Th2.

Effect of Zinc Sulfate and Zinc Glycine Chelate on Concentrations of Acute Phase Proteins in Chicken Serum and Liver Tissue.

The aim of the study was to determine how inorganic and organic forms of zinc affect the concentrations of C-reactive protein (CRP), serum amyloid A (SAA), alpha-1-acid glycoprotein (α-1-AGP), haptoglobin (Hp), and transferrin (TRF) in the blood and liver tissue of 450 1-day-old Ross 308 chicken. Four experimental groups received one the following: inorganic zinc (ZnSO4), a zinc phytase enzyme supplement (ZnSO4-F), organic zinc in combination with glycine (Zn-Gly), or organic zinc supplemented with phytase (Zn-Gly-F). The chicken serum and liver homogenates were assayed using an ELISA kit. The results of the study showed statistically significantly higher serum and liver concentration of SAA in the group of birds that received zinc sulfate in comparison to the group of birds receiving zinc in organic form. A statistically significantly higher serum concentration of CRP and α-1-AGP was also noted in the group receiving zinc sulfate as compared to the Zn-Gly group. Comparison of the serum concentration of TRF between the supplemented groups showed a statistically significant increase in this parameter in the Zn-Gly-F group as compared to the ZSO4-F group. The increase in the serum concentration of Hp in all groups in comparison to the control may indicate stimulation of local immune mechanisms. The results of this study showed an increase in the concentrations of APPs such as AGP and TRF following the administration of zinc glycine chelates, which may demonstrate their effect on metabolic processes in the liver and on immunocompetent cells that regulate the intensity of the immune response.

Effect of the EM Bokashi® Multimicrobial Probiotic Preparation on the Non-specific Immune Response in Pigs.

The aim of the study was to determine the effect of EM Bokashi® on the phagocytic activity of monocytes and granulocytes, oxidative burst, SWC3, and CD11b + CD18+ expression on monocytes and granulocytes, and the serum concentration of cytokine and lysozyme in pig. 60 Sixty female piglets were divided into two groups: I - control and II - experimental. For the experimental group, a probiotic in the form of the preparation EM Bokashi® was added to the basal feed. Flow cytometry was used to determine selected non-specific immune response parameters, intracellular production of hydrogen peroxide by peripheral granulocytes and monocytes, and surface particles in peripheral blood. The EM Bokashi® preparation used in the study was found to increase phagocytic activity mainly in monocytes, with an increased percentage of phagocytic cells in the experimental group. The highest serum lysozyme concentration in the piglets in the experimental group (2.89 mg/dl), was noted on day 42 of the study. In the group of pigs receiving EM Bokashi®, the percentage of phagocytic cells with SWC3 (monocyte/granulocyte) expression was statistically significantly higher than in the control. The increase in the number of cells with SWC3 (monocyte/granulocyte) expression in the peripheral circulation in combination with the greater capacity of the cells for phagocytosis and respiratory burst confirms that the non-specific immune response was modulated in the pigs supplemented with EM Bokashi®.

Molecular analysis of the chromosomal 16S rRNA gene and vapA plasmid gene of Polish field strains of R. equi.

Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80-90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection.

Utilization and acceptance of virtual patients in veterinary basic sciences - the vetVIP-project.

Context: In medical and veterinary medical education the use of problem-based and cased-based learning has steadily increased over time. At veterinary faculties, this development has mainly been evident in the clinical phase of the veterinary education. Therefore, a consortium of teachers of biochemistry and physiology together with technical and didactical experts launched the EU-funded project "vetVIP", to create and implement veterinary virtual patients and problems for basic science instruction. In this study the implementation and utilization of virtual patients occurred at the veterinary faculties in Budapest, Hannover and Lublin. Methods: This report describes the investigation of the utilization and acceptance of students studying veterinary basic sciences using optional online learning material concurrently to regular biochemistry and physiology didactic instruction. The reaction of students towards this offer of clinical case-based learning in basic sciences was analysed using quantitative and qualitative data. Quantitative data were collected automatically within the chosen software-system CASUS as user-log-files. Responses regarding the quality of the virtual patients were obtained using an online questionnaire. Furthermore, subjective evaluation by authors was performed using a focus group discussion and an online questionnaire. Results: Implementation as well as usage and acceptance varied between the three participating locations. High approval was documented in Hannover and Lublin based upon the high proportion of voluntary students (>70%) using optional virtual patients. However, in Budapest the participation rate was below 1%. Due to utilization, students seem to prefer virtual patients and problems created in their native language and developed at their own university. In addition, the statement that assessment drives learning was supported by the observation that peak utilization was just prior to summative examinations. Conclusion: Veterinary virtual patients in basic sciences can be introduced and used for the presentation of integrative clinical case scenarios. Student post-course comments also supported the conclusion that overall the virtual cases increased their motivation for learning veterinary basic sciences.

The effect of feed supplementation with effective microorganisms (EM) on pro- and anti-inflammatory cytokine concentrations in pigs.

Effective microorganisms (EM) are used in numerous fields associated with agriculture. The beneficial effects of EM on the general health of pigs and on production parameters are also determined by the influence of these microbes on immunity. The aim of the study was to assess the effect of feed supplementation with EM on the concentration of pro- and anti-inflammatory cytokines in the serum and in a culture of PBMCs with and without ConA stimulation in pigs. ELISA kits were used to determine the cytokine levels in the porcine serum and the PBMC culture supernatants. Evaluation of the serum concentration of cytokines revealed statistically significantly higher concentrations of IL-2, IL-4, IL-10, IFN-γ and TNF-α in the pigs receiving EM Bokashi. The highest concentration of TGF-ß in the experimental group was noted on the final test day. Evaluation of cytokine concentrations in the PBMC cultures revealed statistically significantly higher concentrations of IL-2, IL-4 and IL-10 from the third day of the experiment. Statistically significantly higher concentrations of TNF-α were obtained in the unstimulated PBMC culture on the second test day and in the culture treated with ConA on the second and the third test days. The results of our study indicate that supplementation of pig feed with EM Bokashi activates the cell-mediated and humoral immune response, ensuring that Th1/Th2 balance is maintained and enhancing immune processes protecting the body against infection.

The effect of feed supplementation with Zakarpacki zeolite (clinoptilolite) on percentages of T and B lymphocytes and cytokine concentrations in poultry.

The available literature lacks information on the effect of Zakarpacki zeolite (clinoptilolite) on the immune system of poultry. Therefore, the aim of this study was to determine the effect of this zeolite on selected indicators of the immune response in poultry by evaluating the expression of cluster of differentiation (CD) surface molecules on T and B lymphocytes and the concentration of IL-2 and IL-10 in the blood. Ninety one-day-old Ross 308 broiler chicks were used in the study. The birds were divided into 3 groups of 30 each. The same basic diet was used in all groups, but groups II and III received a feed additive in the form of 2% and 3% zeolite. Blood samples were collected from all birds on the 40th day of observations. Weight gain in the birds in both experimental groups was significantly higher, and no clinical symptoms of disease were observed. The percentage of CD4+CD25+ T and B lymphocytes was higher in both groups receiving zeolite, but the percentage of CD8+CD25+ T lymphocytes was higher only in the group receiving 3% zeolite. There were no differences between the groups in the percentage of cells with CD3+ and MHC Class II expression. Higher serum concentrations of IL-2 and IL-10 were noted only in group III. The use of zeolites enhances antigen presentation and leads to increased Th1 and Th2 response. Excessive supply of zeolite in the feed leads to a local inflammatory response, which may cause damage to the intestinal barrier.

The effect of feed supplementation with zinc chelate and zinc sulphate on selected humoral and cell-mediated immune parameters and cytokine concentration in broiler chickens.

The ability of poultry to withstand infectious disease caused by bacteria, viruses or protozoa depends upon the integrity of the immune system. Zinc is important for proper functioning of heterophils, mononuclear phagocytes and T lymphocytes. Numerous data indicate that the demand for zinc in poultry is not met in Poland due to its low content in feeds of vegetable origin. The aim of the study was to determine the effect of supplementation of inorganic (ZnSO4 and ZnSO4+ phytase enzyme), and organic forms of zinc (Zn with glycine and Zn with glycine and phytase enzyme) on selected parameters of the cellular and humoral immune response in broiler chickens by evaluating the percentage of CD3+CD4+, CD3+CD8+, CD25+, MHC Class II, and BU-1+ lymphocytes, the phagocytic activity of monocytes and heterophils, and the concentration of IL-2, IL-10 and TNF-α in the peripheral blood. Flow cytometry was used to determine selected cell-mediated immune response parameters. Phagocytic activity in whole blood was performed using the commercial Phagotest kit (ORPEGEN-Pharma, Immuniq, Poland). The results showed that supplementation with zinc chelates causes activation of the cellular and humoral immune response in poultry, helping to maintain the balance between the Th1 and Th2 response and enhancing resistance to infections. In contrast with chelates, the use of zinc in the form of sulphates has no immunomodulatory effect and may contribute to the development of local inflammatory processes in the digestive tract, increasing susceptibility to infection.

Quality of fresh and chilled-stored raccoon dog semen and its impact on artificial insemination efficiency.

BACKGROUND: The aim of the study was to evaluate the quality of fresh raccoon dog semen and raccoon dog semen stored at 4 °C. The qualitative evaluation was based on apoptosis in the sperm cells, which was tested by the Annexin V/Pi assay, the TUNEL method and JC-1. In addition, the suitability of the semen for insemination and its effect on reproduction in females were determined in relation to the time of storage. RESULTS: During cold storage of the semen, in the samples from all groups a gradual decrease was noted in the percentage of live cells, and an increase in the percentage of cells with abnormal morphology, exhibiting changes typical of late-stage apoptosis (V+/PI+), and of necrotic cells (V-/PI+). There was a significant increase in the percentage of ApoBrDu + sperm cells, while the mitochondrial membrane potential of the sperm decreased significantly after 12 h of storage at 4 °C in the case of lower-quality semen and after 48 h in the case of semen of good quality. As the percentage of sperm with DNA and cell membrane damage increased and the mitochondrial membrane potential decreased, there was an increase in AspAT and acrosin activity. The increase in the percentage of apoptotic sperm in the raccoon dog semen stored at 4 °C resulted in a decrease in the number of females with cubs. CONCLUSIONS: Identification of apoptotic changes in sperm by flow cytometry using the annexin assay, the TUNEL assay and evaluation of mitochondrial membrane potential can be recommended for determination of the suitability of raccoon dog semen for artificial insemination. The study shows that fresh raccoon dog semen should not be used for insemination more than 48 h after collection in the case of semen of very high quality, or after more than 24 h in the case of semen of poorer quality. Cytometric methods of semen analysis should also be used to evaluate various extenders of raccoon dog semen and methods of cryopreservation in terms of ensuring sperm viability, fertilization capacity, and suitability for insemination.

Effects of feed supplementation with glycine chelate and iron sulfate on selected parameters of cell-mediated immune response in broiler chickens.

Because little is known about the impact of chelated (Fe-Gly, Fe-Gly+F) and inorganic (FeSO4, FeSO4+F) iron products on immune response parameters in broiler chickens, the objective of the study was to determine the effects of inorganic and organic forms of iron on selected parameters of the cell-mediated immune response in broiler chickens by assessing the percentage of CD3(+)CD4(+), CD3(+)CD8(+), CD25(+), and MHC Class II lymphocytes, as well as the CD4(+)/CD8(+) ratio and IL-2 concentration in the peripheral blood. The experiments were conducted using 50day-old Ross 308 roosters. The test material was peripheral blood. Flow cytometry was used to determine selected cell-mediated immune response parameters. The results obtained indicate that the use of iron chelates in the diet of broiler chickens may stimulate cellular defense mechanisms. As a result of the experiment an increase was observed in the percentage of Th1, mainly T CD4(+) and T CD8(+). It was also noted that application of chelated iron can increase production of T CD8(+) cytotoxic cells and IL-2, which promotes the body's natural response to developing inflammation. There were no changes in T CD4(+), T CD8(+), T CD25(+) or MHC II lymphocyte subpopulations in the chickens following application of the inorganic form of iron.

Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland.

Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15-17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85-90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.

Phase I/II clinical trial of encapsulated, cytochrome P450 expressing cells as local activators of cyclophosphamide to treat spontaneous canine tumours.

Based upon promising preclinical studies, a clinical trial was performed in which encapsulated cells overexpressing cytochrome P450 enzyme isoform 2B1 were implanted around malignant mammary tumours arising spontaneously in dogs. The dogs were then given cyclophosphamide, one of the standard chemotherapeutic agents used for the treatment of mammary tumours. The dogs were assessed for a number of clinical parameters as well as for reduction in tumour size. The treatment was well tolerated with no evidence of adverse reactions or side effects being associated with the administration of the encapsulated cells. Reductions in tumour size of more than 50% were observed for 6 out of the 11 tumours analysed while 5 tumours showing minor responses, i.e. stable disease. In contrast, the tumours that received cyclophosphamide alone showed only stable disease. Taken together, this data suggests that encapsulated cytochrome P450 expressing cells combined with chemotherapy may be useful in the local treatment of a number of dog mammary tumours and support the performance of further clinical studies to evaluate this new treatment.

Genetic typing of equine influenza virus isolated in Poland in 2005 and 2006.

Two equine influenza virus strains were isolated from horses during the local respiratory disease outbreaks in Poland in 2005 and 2006. The H3 equine influenza viral RNA was amplified directly from the clinical specimens with RT-PCR and HA1 fragments were sequenced. The highest homology of HA1 nucleotide sequences of A/eq/Pulawy/05 with A/eq/Aboyne/1/05 and A/eq/Pulawy/06 with A/eq/Essex/2/05 was found. The phylogenetic analysis based on amino acid sequences of HA1 fragments of 84 equine influenza virus strains isolated in Europe during the period of 1976-2007 was conducted to determine the evolutionary relationship of the two Polish and the other European isolates. The resulting phylogenetic tree clearly clustered A/eq/Pulawy/05 with the strains belonging to the European lineage of the equine influenza virus. On the other hand A/eq/Pulawy/06 was placed in the Florida sub-lineage of the American type strains. The presence of the same amino acids: methionine, asparagine and threonine at the positions 48, 159 and 163 respectively, in both Polish isolates, despite the fact that the strains are grouped in two different lineages may indicate the existence of the common ancestor. It is possible that A/eq/Pulawy/06 evolved locally rather than was introduced.