The effect of Limosilactobacillus fermentum 2i3 and 0.6% addition of humic substances on production parameters and the immune system of broilers.

The widespread use of antibiotics in the poultry industry as growth promoters has led to the emergence of bacterial resistance, which poses a significant health risk to humans and animals. Substances of natural origin, such as probiotic bacteria and humic substances, can be a promising solution. The aim of this experiment was to study the effect of the administration of a probiotic strain of Limosilactobacillus fermentum 2i3 and/or a new formula of humic substances specifically designed for detoxification on the production parameters, including gene expression of myogenic growth factors and selected parameters of the immune response. We found that production parameters such as feed conversion ratio and weekly weight gain, as well as gene expression of mucin-2 and immunoglobulin A, were positively influenced mainly by the administration of L. fermentum 2i3. Similarly, the percentage of active phagocytes and their absorption capacity as well as the proportions of CD8+ and CD4+CD8+ T-lymphocyte subpopulations were significantly increased. The addition of humic substances, either alone or in combination with probiotics, significantly reduced the aforementioned parameters compared to the control. On the other hand, the relative gene expression for all myogenic growth factors was the highest in the humic group alone. Based on the results obtained, we can confirm the immunostimulating effect of L. fermentum 2i3 administered in drinking water, which also had an impact on important production parameters of broiler meat. On the other hand, in the combined group there was no expected potentiation of the positive effects on the observed parameters.

Differential Activity of Human Leukocyte Extract on Systemic Immune Response and Cyst Growth in Mice with Echinococcus Multilocularis Infection After Oral, Subcutaneous and Intraperitoneal Routes of Administration.

Alveolar echinococcosis (AE) caused by the larval stage of Echinococcus multilocularis is serious parasitic diseases associated with the host´s immunosuppression. The effects of human non-immune dialyzable leukocyte extract (DLE) on immune cells in blood and spleen and parasitic cysts weight in Balb/c mice after oral (PO), subcutaneous (SC) and intraperitoneal administration (IP) were compared. The reduction in cysts weight (p < 0.01) was recorded after PO route, whereas moderate reduction was found after SC and IP routes. The elevation of lymphoid populations in blood and spleen was found after PO administration (p < 0.01) in parallel with reduced myeloid population. Infection-elicited decline in B220+B cells was partially abolished by PO route, but DLE routes did not influence the CD3+ T cells. The proportions of CD3+CD4+Th lymphocytes were moderately upregulated, whereas CD3+CD8+Tc populations were reduced after all DLE routes (p < 0.01). PO administration increased CD11b+MHCIIhigh blood monocytes, CD11b-SigleF+ cell, but not CD11b+Si-glecF+ eosinophils in the blood, stimulated after SC and IP routes. DLE induced downregulation of NO production by LPS-stimulated adherent splenocytes ex vivo. Con A-triggered T lymphocyte proliferation was associated with the elevated IFN-γ production and transcription factor Tbet mRNA expression. The alleviation of Th2 (IL-4) and Treg (TGF-ß) cytokine production by lymphocytes ex vivo paralleled with downregulation of gene transcription for cytokines, GATA and FoxP3. Reduction of myeloid cells with suppressive activity was found. The SC and IP routes affected partially the cysts weights, diminished significantly gene transcription, NO levels and Th2 and Treg cytokines production. Results showed that PO route of DLE administration was the most effective in ameliorating immunosuppression via stimulation of Th1 type, reducing Th2 and Treg type of immunity and CD3+CD8+Tc lymphocytes in the blood and spleens during E. multilocularis infection in mice.

Antiparasitic Effects of Selected Isoflavones on Flatworms.

Medicinal plants have been successfully used in the ethno medicine for a wide range of diseases since ancient times. The research on natural products has allowed the discovery of biologically relevant compounds inspired by plant secondary metabolites, what contributed to the development of many chemotherapeutic drugs. Flavonoids represent a group of therapeutically very effective plant secondary metabolites and selected molecules were shown to exert also antiparasitic activity. This work summarizes the recent knowledge generated within past three decades about potential parasitocidal activities of several flavonoids with different chemical structures, particularly on medically important flatworms such as Schistosoma spp., Fasciola spp., Echinococcus spp., Raillietina spp., and model cestode Mesocestoides vogae. Here we focus on curcumin, genistein, quercetin and silymarin complex of flavonolignans. All of them possess a whole spectrum of biological activities on eukaryotic cells which have multi-therapeutic effects in various diseases. In vitro they can induce profound alterations in the tegumental architecture and its functions as well as their activity can significantly modulate or damage worm´s metabolism directly by interaction with enzymes or signaling molecules in dose-dependent manner. Moreover, they seem to differentially regulate the RNA activity in numbers of worm´s genes. This review suggests that examined flavonoids and their derivates are promising molecules for antiparasitic drug research. Due to lack of toxicity, isoflavons could be used directly for therapy, or as adjuvant therapy for diseases caused by medically important cestodes and trematodes.

Effect of fungal solid-state fermented product enriched with gamma-linolenic acid and ß-carotene on blood biochemistry and immunology of broiler chickens.

The aim of this study was to investigate the effect of the addition of fungal solid-state fer-mented product (FP) enriched with gamma-linolenic acid (GLA) and ß-carotene to feed on the haematological and immunological parameters of broiler chickens. Eighty 1-day-old COBB 500 broiler chickens were divided into two groups. The control group was fed with basic diets and chickens of the experimental group received 10% addition of FP, while the amount of basic diet was reduced. FP was produced during a solid-state fermentation (SSF) process using Umbellopsis isabellina CCF2412 as a producer of GLA and ß-carotene. After 38 days of feeding, blood sam-ples were collected and analyzed. Lower total and LDL-cholesterol values were measured in blood samples of the experimental animals (p⟨0.05). However, the triacylglycerol content was higher in the experimental group (p⟨0.05). Significantly higher levels of hematocrit and hemo-globin, and lower eosinophil and basophil content in the experimental group were recorded (p⟨0.05). The experimental group showed higher numbers of B lymphocytes and greater phago-cytic capacity (p⟨0.05). The results indicate that a fermented product produced by SSF, using the fungal strain Umbellopsis isabellina, is a good source of GLA and ß-carotene, which can influence the biochemical, hematological and immunological parameters of broiler chickens.

Differential Sensitivity of Myeloid and Lymphoid Cell Populations to Apoptosis in Peritoneal Cavity of Mice with Model Larval Mesocestoides Vogae Infection.

The metacestode stage of the tapeworm Mesocestoides vogae (M. vogae) has the ability of asexual growth in the peritoneal cavity of rodents and other intermediate hosts without restriction. Early immunological events have decisive role in the establishment of infection. In the present study we investigated the kinetic of myeloid and lymphoid cell populations and the proportions of cells undergoing apoptosis in peritoneal cavities of mice within the first month after oral infection with M. vogae larvae. Proportions of cell phenotypes and apoptotic cells were examined by flow cytometry and by microscopical analysis of cells following May/Grünwald staining and fluorescent stain Hoechst 33234, respectively. Total numbers of peritoneal cells increased and their distribution changed towards accumulation of myelo-monocytic cell lineage in the account of reduced proportions of lymphoid cells. CD4+ T cell subpopulations were more abundant than CD8+ and their proportions elevated within two weeks post infection (p.i.) which was followed by a significant decline. Expression level of CD11c marker on myelo-monocytic cells revealed phenotype heterogeneity and proportions of cells with low and medium expression elevated from day 14 p.i. along with concurrent very low presence of CD11chigh phenotype. Lymphoid cell population was highly resistant to apoptosis but elevated proportions of myeloid cells were in early/late stage of apoptosis. Apoptosis was detected in a higher number of adherent cells from day 14 p.i. onwards as evidenced by nuclear fluorescent staining. By contrast, cells adherent to larvae, mostly macrophages and eosinophils, did not have fragmented nuclei. Our data demonstrated that apoptosis did not account for diminished population of peritoneal lymphoid cells and substantial proportions of myeloid cells seem to be more susceptible to apoptotic turnover in peritoneal cavity of mice with ongoing M. vogae infection, suggesting their important role in the host-parasite interactions.

Systemic immune response of gnotobiotic mice infected with porcine circovirus type 2 after administration of Lactobacillus reuteri L26 Biocenol™.

In our previous study we confirmed an antiviral activity of probiotic Lactobacillus reuteri L26 which was mediated by stimulation of local intestinal immunity. The aim of this paper was to evaluate the influence of L. reuteri L26 on the systemic immune response in gnotobiotic mice infected with porcine circovirus type 2 (PCV2). A total of 30 germ-free mice were divided into 3 groups and animals in noninfected and infected control groups (NC and IC; n=10) received sterile de Man-Rogosa-Sharpe broth for 7 days and animals in experimental group L+PCV (n=10) were inoculated with L. reuteri L26. Subsequently, mice in L+PCV and IC groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). The results showed an increase of percentage of cytotoxic cells (CD8+ and CD49b+CD8-) and oxidative burst of phagocytes, up-regulation of the gene expression of RANTES, granulocyte-macrophage colony-stimulating factor, interferon-γ and immunoglobulin A in blood above all in the later phase of infection (14 dpi) in L+PCV group accompanied by higher load of PCV2 in the serum. These findings indicate that L. reuteri L26 has a potential to induce systemic immune reaction, but in gnotobiotic mice immune stimulation can increase virus replication.

Effect of fungal gamma-linolenic acid and beta-carotene containing prefermented feed on immunity and gut of broiler chicken.

Gamma-linolenic acid (GLA) is a fatty acid from the ω-6 family. It is able to deliver a wide range of health benefits arising from its anti-inflammatory effects. An insufficient supply of GLA from agricultural and animal sources resulted in the development of a fermentation technique using lower filamentous fungi, which have the ability to accumulate high concentrations of GLA and beta-carotene during solid-state fermentation of cereals. The goal of this study was to observe the influence of the addition of prefermented cereal product, containing high amounts of GLA and beta-carotene, into the feed of broiler chickens on their immune status, and also the number of lactic acid bacteria and enterobacteria in gut content, which has never been studied before. Immunostimulation in the GLA group was manifested by a significant increase in the oxidative burst of phagocytes, CD4+CD8- lymphocytes in blood, and the CD4: CD8 ratio. Upregulation of gene expression for IgA in the GLA group indicates that the B-lymphocytes were stimulated at a local gut level. In the caecum, increased mRNA expression for mucin-2 and insulin-like growth factor was observed in the GLA group, which could contribute mainly to the protection of the intestinal mucosa and to better growth and regeneration of skeletal muscles. Improved immune activation and protection of the intestinal mucosa were subsequently reflected in a change of the microbial composition in gut contents; a significant reduction of enterobacteria occurred after GLA administration. We can conclude that prefermented cereals containing fungal GLA and beta-carotene represent a low-cost supplement for broiler diet having a beneficial health effect.

Influence of Lactobacillus reuteri L26 Biocenol™ on immune response against porcine circovirus type 2 infection in germ-free mice.

Probiotic bacteria are frequently used for prevention of bacterial infections of the gastrointestinal tract, but there are only limited studies on their efficacy against viral gut infections in animals. The aim of this study was to investigate the effect of probiotic Lactobacillus reuteri L26 BiocenolTM on the innate and adaptive immune responses in germ-free Balb/c mice, experimentally infected by porcine circovirus type 2 (PCV2), which confers immunosuppressive effect. A total of 30 six-week-old female mice were divided into 3 groups and animals in experimental group LPCV (n=10) were inoculated with L. reuteri L26, animals in the control group (C; n=10) and experimental group PCV (n=10) received sterile De Man-Rogosa-Sharpe broth for 7 days. Subsequently, mice from both experimental groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). Virus load in faeces, ileum and mesenteric lymph nodes (MLN); as well as gene expression of selected cytokines, immunoglobulin A (IgA) and polymeric Ig receptor (PIgR) in the ileum, and percentage of CD8+, CD19+ and CD49b+CD8- cells in the MLN were evaluated. Our results showed that L. reuteri significantly decreased the amount of PCV2 in faeces and in the ileum, and up-regulated the gene expression of chemokines, interferon (IFN)-γ, IgA and PIgR in the ileum. Increased IFN-γ mRNA level was accompanied by higher proportion of natural killer cells and up-regulated IgA and PIgR gene expressions were in accordance with significantly higher percentage of CD19+ lymphocytes in the MLN. These findings indicate that probiotic L. reuteri has an antiviral effect on PCV2 in the intestine which is mediated by stimulation of local gut immune response.

Histo-FISH protocol to detect bacterial compositions and biofilms formation in vivo.

The study of biofilm function in vivo in various niches of the gastrointestinal tract (GIT) is rather limited. It is more frequently used in in vitro approaches, as an alternative to the studies focused on formation mechanisms and function of biofilms, which do not represent the actual in vivo complexity of microbial structures. Additionally, in vitro tests can sometimes lead to unreliable results. The goal of this study was to develop a simple approach to detect bacterial populations, particularly Lactobacillus and Bifidobacterium in biofilms, in vivo by the fluorescent in situ hybridisation (FISH) method. We standardised a new Histo-FISH method based on specific fluorochrome labelling probes which are able to detect Lactobacillus spp. and Bifidobacterium spp. within biofilms on the mucosal surface of the GIT embedded in paraffin in histological slices. This method is also suitable for visualisation of bacterial populations in the GIT internal content. Depending on the labelling probes, the Histo-FISH method has the potential to detect other probiotic strains or pathogenic bacteria. This original approach permits us to analyse bacterial colonisation processes as well as biofilm formation in stomach and caecum of BALB/c and germ-free mice.

Flow cytometry as an auxiliary tool for the selection of probiotic bacteria.

Selection of appropriate bacterial strains is crucial for development of new probiotic preparations. The fundamental prerequisite for potential efficacy of a probiotic preparation for oral application is the selection of appropriate bacterial strains with good gastrointestinal colonisation abilities, antimicrobial activity, and tolerance of conditions in the gastrointestinal tract, resistance to different antimicrobial agents, survival during processing and storage. The strain should be genetically stable, it should have good growth properties, to maintain its high viability at processing and when in storage. Mostly, the properties of promising strains are tested in the first phase in vitro, and only the best ones undergo subsequent in vivo testing. in vitro tests are often performed by classical microbiological cultivation methods which are material and time consuming, and they are not able to distinguish between 'viable but nonculturable' and dead bacteria. Flow cytometry is usually used for counting, phenotyping or functional characterisation of immune cells. Nowadays, flow cytometry is increasingly used in microbiology for counting bacteria, determining their viability and metabolic activity, detecting specific strains or testing their adherence abilities. The utilisation of flow cytometry in combination with an appropriate fluorescent labelling represents an effective and rapid method for the selection of probiotic bacteria.

Anti-inflammatory and immunoregulatory effects of flax-seed oil and Lactobacillus plantarum - Biocenol™ LP96 in gnotobiotic pigs challenged with enterotoxigenic Escherichia coli.

The aim of this study was to determine the immune response after preventive administration of flax-seed oil (rich in n-3 PUFAs) or probiotic strain Lactobacillus plantarum - Biocenol™ LP96 or their combination in the jejunum of ETEC-challenged gnotobiotic pigs. Subsequently, gene expression of selected cytokines, phagocytic activity of leukocytes from peripheral blood and percentage of CD2(+), CD4(+), CD8(+) and CD4(+)CD25(+) lymphocytes in jejunal mucosa were evaluated. Our results showed that combined treatment down-regulates IL-1α and IL-8 gene expression, up-regulates IFN-γ and tends to regulate inflammation induced by ETEC through cytokine IL-10. In general, changes in cytokine gene expression correlated with the proportions of immune cells isolated from the same part of the jejunal mucosa. Results indicate that probiotic L. plantarum in combination with flax-seed oil rich in n-3 PUFAs has anti-inflammatory properties, stimulates Th1-mediated cell immunity and phagocytosis, and tends to regulate the inflammatory response induced by ETEC.

Multiplex PCR assay for detection of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis in lungs of pigs from a slaughterhouse.

Multiplex PCR has been developed for parallel identification of Actinobacillus pleuropneumoniae, Pasteurella multocida and Haemophilus parasuis, important pathogens of swine, responsible for considerable economic losses in swine industry. Multiplex PCR and bacteriological cultivation was used to analyze lung samples from slaughterhouse pigs. From a total of 219 lung samples, 164 (74.9 %) were positive for P. multocida, 45 (20.5 %) for A. pleuropneumoniae and 4 (1.83 %) for H. parasuis. Bacteriological examination revealed that 145 samples (66.2 %) were positive for P. multocida, 31 (14.2 %) for A. pleuropneumoniae and 2 (0.91 %) for H. parasuis.

Improvement of the probiotic effect of micro-organisms by their combination with maltodextrins, fructo-oligosaccharides and polyunsaturated fatty acids.

Probiotics could represent an effective alternative to the use of synthetic substances in nutrition and medicine. The data concerning the efficacy of probiotics are often contradictory. This paper focuses on the enhancement of the efficacy of probiotics by their combination with synergistically acting components of natural origin. Maltodextrins can be obtained by enzymatic hydrolysis of starch and are suitable for consumption. Administration of Lactobacillus paracasei together with maltodextrin decreased the number of Escherichia coli colonising the jejunal mucosa of gnotobiotic piglets by 1 logarithm compared to the control group. Fructo-oligosaccharides (FOS) are naturally occurring oligosaccharides, mainly of plant origin. L. paracasei administered in combination with FOS significantly increased counts of Lactobacillus spp., Bifidobacterium spp., total anaerobes and total aerobes compared to the control group as well as the L. paracasei group. It also significantly decreased Clostridium and Enterobacterium counts in the faeces of the weanling piglets compared with the control group. Dietary lipids influence the gastrointestinal microbiota and specifically the population of lactic acid bacteria. In gnotobiotic piglets the oral administration of an oil containing polyunsaturated fatty acids (PUFA) significantly increased the number of L. paracasei adhering to jejunal mucosa compared to the control group. Our results showed that maltodextrin KMS X-70 and PUFA can be used to enhance the effect of probiotic micro-organisms in the small intestine, and similarly FOS enhance the effect of probiotic micro-organisms in the large intestine.