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

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

ABSTRACT: 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

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

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

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

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

Influence of Effective Microorganisms and Clinoptilolite on Gut Barrier Function, Intestinal Health and Performance of Broiler Chickens during Induced <i>Eimeria tenella</i> Infection

The prohibition of certain coccidiostats in poultry has created a need to seek an alternative to control Eimeria infection. The aim of this study was to evaluate the effects of effective microorganisms (EM) in a multi-strain probiotic (Bokashi®), with clinoptilolite as a feed supplement on the mRNA expression of tight junction proteins and redox enzymes in the caecal tissue of chickens infected with E. tenella. The integrity of the intestinal barrier was tested by determining the concentration of fluorescein isothiocyanate dextran (FITC-d) in the chicken’s serum. A total of 600 1-day-old Ross 308 male chickens received diets with a 0.5% or 0.8% concentration of the probiotic together with clinoptilolite. The experiment used 5 treatment groups, and a control group, each with 5 replicates with 20 birds. The results indicate that the use of the 8 kg/t of feed multi-strain probiotic together with clinoptilolite in the diet of poultry caused a significant reduction in the number of E. tenella oocysts in the faeces and caecum and significantly improved the growth rate of chicken broilers infected with E. tenella. In addition, the probiotic and clinoptilolite enhanced antioxidant processes in the caecal mucosa and reduced oxidative stress induced by E. tenella infection

Plasmid profiles of <i>R</i>. <i>equi</i> isolates, clinical and environmental strains.

<p>Plasmid profiles of <i>R</i>. <i>equi</i> isolates, clinical and environmental strains.</p

Dynamics of tumour size change in the study group expressed as a percentage of initial tumour size.

<p>Dynamics of tumour size change in the study group expressed as a percentage of initial tumour size.</p

The Effect of Glutamine as Feed Additive on Selected Parameters of the Nonspecific Immune Response in Pigs

The use of feed additives containing glutamine can influence the growth and development of piglets during the weaning period. The aim of this study was to determine the effect of feed supplementation with 0.5% L-glutamine on selected parameters of the nonspecific immune response of pigs. The research was carried out on 60 pigs (Polish Large White × Polish Landrace), from 28 days of age to slaughter. The obtained results showed an increased percentage of phagocytic cells (monocytes and granulocytes) and oxygen blast cells in pigs between 28 and 70 days of age, proving that non-specific immune mechanisms were stimulated, which contributed to the improvement of the processes of antigen elimination from the body. The increase in the percentage of cells expressing SWC3, CD11b/CD18+, CD14+ and CD14+CD16+ molecules on granulocytes and monocytes during this period resulted in the enhancement of the host defense mechanisms by stimulating phagocytosis and enhancing the mechanisms of a non-specific immune response. The high concentration of TNF-α and IL-1β as well as Il-10 in the experimental group indicates the cellular phenotype of the Th1-type response, and the maintenance of the immune balance between the pro-inflammatory and anti-inflammatory responses and ensuring the homeostasis of the organism