BACKGROUND: In view of the ever-increasing representation of Staphylococcus spp. strains resistant to various antibiotics, the development of in vivo models for evaluation of novel antimicrobials is of utmost importance. METHODS: In this article, we describe the development of a fully immunocompetent porcine model of extensive skin and soft tissue damage suitable for testing topical antimicrobial agents that matches the real clinical situation. The model was developed in three consecutive stages with protocols for each stage amended based on the results of the previous one. RESULTS: In the final model, 10 excisions of the skin and underlying soft tissue were created in each pig under general anesthesia, with additional incisions to the fascia performed at the base of the defects and immediately inoculated with Staphylococcus aureus suspension. One pig was not inoculated and used as the negative control. Subsequently, the bandages were changed on Days 4, 8, 11, and 15. At these time points, a filter paper imprint technique (FPIT) was made from each wound for semi-quantitative microbiological evaluation. Tissue samples from the base of the wound together with the adjacent intact tissue of three randomly selected defects of each pig were taken for microbiological, histopathological, and molecular-biological examination. The infection with the inoculated S. aureus strains was sufficient during the whole experiment as confirmed by both FPIT and from tissue samples. The dynamics of the inflammatory markers and clinical signs of infection are also described. CONCLUSIONS: A successfully developed porcine model is suitable for in vivo testing of novel short-acting topical antimicrobial agents.

• Keywords
• Staphylococcus aureus, animal model, antimicrobial agents, porcine model, skin and soft‐tissue infection (SSTI), wound infection,
• MeSH
• Anti-Bacterial Agents * administration & dosage   therapeutic use   pharmacology   MeSH
• Administration, Topical MeSH
• Soft Tissue Infections * drug therapy   microbiology   MeSH
• Skin microbiology   pathology   MeSH
• Methicillin-Resistant Staphylococcus aureus * drug effects   MeSH
• Disease Models, Animal * MeSH
• Swine MeSH
• Staphylococcal Skin Infections * drug therapy   microbiology   MeSH
• Staphylococcal Infections * drug therapy   microbiology   MeSH
• Staphylococcus aureus * drug effects   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH

Using probiotics represents a potential solution to post-weaning diarrheal diseases in piglets on commercial farms. The gastrointestinal tract of wild boars serves as a promising reservoir of novel lactic acid bacteria with suitable probiotic characteristics. In this study, we isolated eight bacterial strains from the intestinal content of wild boars identified as representatives of the species Bifidobacterium apri, Lactobacillus amylovorus, and Ligilactobacillus salivarius. These isolates underwent in vitro analysis and characterisation to assess their biological safety and probiotic properties. Analysis of their full genome sequences revealed the absence of horizontally transferrable genes for antibiotic resistance. However, seven out of eight isolates harboured genes encoding various types of bacteriocins in their genomes, and bacteriocin production was further confirmed by mass spectrometry analysis. Most of the tested strains demonstrated the ability to inhibit the growth of selected pathogenic bacteria, produce exopolysaccharides, and stimulate the expression of interleukin-10 in porcine macrophages. These characteristics deem the isolates characterised in this study as potential candidates for use as probiotics for piglets during the post-weaning period.

• Keywords
• antibiotic susceptibility, antimicrobial activity, bacteriocins, exopolysaccharides, interleukin-10,
• Publication type
• Journal Article MeSH

The aim of this study was to establish a cell culture system for the generation of porcine monocyte-derived macrophages (MDMs) under reduced-serum conditions. Cultures based on either the Nu-Serum™ Growth Medium Supplement (NUS) or a conventional fetal bovine serum (FBS) were compared, which included the assessment of FBS from two different providers (FBS1 and FBS2). The data obtained confirmed the significant impact of culture conditions on in vitro-generated MDMs. The MDMs cultured under reduced-serum conditions showed increased levels of IL-1β and CD86 mRNA and a proinflammatory cytokine profile, characterized by the increased mRNA expression of IL-23p19, CXCL10, and CCL5. Phagocytic and respiratory burst activities were not adversely affected. Surprisingly, the difference between the two FBSs was much more pronounced than the effect of the reduced-serum supplement. The FBS1 culture conditions gave rise to macrophages with higher surface levels of CD14, CD16, and CD163, a lower CD80 mRNA expression, and an increased induction of IL-10 gene expression. In contrast, none of these trends were observed in macrophage cultures supplemented with FBS2. Instead, the FBS2 culture showed increased levels of IL-1b and CD86 mRNA. In conclusion, reduced-serum culture is a useful tool for in vitro porcine MDM generation, in line with the current research trend of reducing FBS use in biological research.

• Keywords
• in vitro, monocyte-derived macrophages, pig, porcine, serum reduction,
• Publication type
• Journal Article MeSH

In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

• Keywords
• Glaesserela parasuis, Haemophilus parasuis, antibodies, antioxidants, capsule, catalase, porcine alveolar macrophages, reactive oxygen species,
• MeSH
• Macrophages, Alveolar immunology   metabolism   microbiology   MeSH
• Antigens, Bacterial immunology   MeSH
• Bacterial Capsules immunology   MeSH
• Phagocytosis MeSH
• Haemophilus parasuis immunology   pathogenicity   MeSH
• Haemophilus Infections immunology   metabolism   microbiology   MeSH
• Kinetics MeSH
• Cells, Cultured MeSH
• Antibodies, Neutralizing immunology   metabolism   MeSH
• Antibodies, Bacterial immunology   metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Serogroup MeSH
• Antibody Specificity MeSH
• Sus scrofa MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Bacterial MeSH
• Antibodies, Neutralizing MeSH
• Antibodies, Bacterial MeSH
• Reactive Oxygen Species MeSH

BACKGROUND: Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. RESULTS: Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. CONCLUSION: Our results suggest that LF's ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.

• Keywords
• Inflammatory cytokines, LPS, NFκB, NIK, TLR4,
• MeSH
• Cytokines genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Lactoferrin isolation & purification   pharmacology   MeSH
• Humans MeSH
• Lipopolysaccharides pharmacology   MeSH
• Macrophages drug effects   MeSH
• Swine MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Gene Expression Regulation drug effects   MeSH
• Signal Transduction drug effects   MeSH
• Toll-Like Receptor 4 genetics   metabolism   MeSH
• Protein Binding MeSH
• Inflammation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokines MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Lactoferrin MeSH
• Lipopolysaccharides MeSH
• Nik related kinase MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Toll-Like Receptor 4 MeSH

BACKGROUND: This study aims to investigate the anti-inflammatory effect of biologically active phospholipids (BAP) used in preparations for clinical practice in humans. Until date, except anti-neoplastic ability, little is known about anti-inflammatory property of the phospholipids. METHODS: While the course of bacterially induced acute pneumonia and markers of inflammation were studied in in vivo system in pigs orally supplemented with BAP, the pro- and anti-inflammatory response of lipopolysaccharide-stimulated porcine monocyte-derived macrophages to 24 h- and 48 h-treatment by BAP was investigated in in vitro system. In vivo, the animal health status was monitored and pro-inflammatory IL-1β and IL-8 in sera were detected by ELISA during the experiment, while bronchoalveolar lavage fluids (BALF) and the lungs were examined post-mortem. Total and differential counts of white blood cell (WBC) were determined in blood and BALF. In vitro, mRNA expression of pro-inflammatory (TNF-α, IL-1β, CXCL10) and anti-inflammatory (IL-10 and Arg1) cytokines, and level of activated caspase 1 and phosphorylated protein kinase C epsilon (pPKCϵ), were studied using qRT-PCR and Western blot, respectively. For the purposes of both systems, 6 animals were used in each of the BAP-supplemented and the control groups. RESULTS: In vivo, BAP had a positive influence on the course of the disease. The immunomodulatory effects of BAP were confirmed by lower levels of IL-1β, IL-8, and a lower WBC count in the supplemented group in comparison with the control group. A lower percentage of lung parenchyma was affected in the supplemented group comparing to the control group (on average, 4% and 34% of tissue, respectively). In vitro, BAP suppressed mRNA expression of mRNA for IL-10 and all pro-inflammatory cytokines tested. This down-regulation was dose- and time-dependent. Arg1 mRNA expression remained unaffected. Further dose- and time-dependent suppression of the activated caspase 1 and pPKCϵ was detected in macrophages when treated with BAP. CONCLUSIONS: Our results demonstrate that BAP has anti-inflammatory and immunomodulatory properties, thus emphasizing the potential of this compound as a natural healing agent.

• MeSH
• Anti-Inflammatory Agents pharmacology   MeSH
• Pneumonia, Bacterial metabolism   pathology   MeSH
• Bronchoalveolar Lavage Fluid cytology   MeSH
• Cytokines blood   MeSH
• Phospholipid Ethers pharmacology   MeSH
• Cells, Cultured MeSH
• Leukocytes MeSH
• Lipopolysaccharides MeSH
• Macrophages drug effects   MeSH
• Lung drug effects   pathology   MeSH
• Swine MeSH
• Inflammation drug therapy   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Inflammatory Agents MeSH
• Cytokines MeSH
• Phospholipid Ethers MeSH
• Lipopolysaccharides MeSH