PLANT SPECIES’ DEPENDANT IN VITRO IMMUNE RESPONSE AND CHANGES IN THE PORTED BACTERIOME IN SMALL VERSUS LARGE RUMINANTS

Abstract Low-input farms, where cohabitation of various species is frequent, ensure welfare by providing outdoor free-roaming and also offering the opportunity for expression of physiological behaviour of the animals. The study aimed at investigating the bacteriome and the immune potential of cohabitants (bovine and sheep) from a low input farm. The research was carried out on nasal swabs and blood samples from bovine (n=7), and sheep (n=17) cohabiting on the same low-input farm. The swabs were subjected to clasical bacteriology techniques and bichemical identification by API tests (Bio Merieux France). To monitor the in vitro blast transformation capacity of lymphocytes, aliquots of blood were mixed 1:4 with RPMI1640 (Sigma Aldrich, USA), further divided in 200µl aliquots in duplicate in 96 well-plates and supplemented with a mitogen (PHA), alcohol control and alcoholic extract of Symphytum officinale (1.5 µl/well). The plates were incubated at 37⁰C for 72 h. The glucose residue was quantified by spectrophotometry (SUMAL PE2, Karl Zeiss, Jena) and blastogenic indices (SI%) were calculated. The groups were compared by Student’s t test for statistical significance of the results. P. aeruginosa, A. hydophila/caviae, E. cloacae, Pasteurella pneumotropica/M. haemolytica, Sphingomonas paucimobilis were isolated from bovine with an average MAR index of 0.288, while P. oryzihabitans, P. aeruginosa, P. fluorescens/P. putida, E. cloacae, Shigella spp. and Ewingella americana with a MAR index of 0.37 were isolated from sheep. The spontaneous SI was higher in cows (55.7± 10.3%) while PHA induced SI was higher in bovine (53.2±9.21%) and sheep (37.81±5.08%). The Symphytum officinale extract did not influence the SI in either bovine or sheep. Considering the similar influential factors acting on cohabiting animals on a low-input farm, there was a species-specific immune system controlled MAR resistance of the bacteriome, which the Symphytum officinale extract could not influence. Key words: bovine, sheep, bacteriome, in vitro cellular response, MAR index, Symphytum officinal

Interdependence of productive effort and in vitro vegetal extract treatment on specific cell-mediated immunity in horses

The research aimed at identifying and rendering alcoholic extracts of various plants based on their in vitro potential in stimulating cell mediated immunity and in alleviating stress effects subsequent to workout type in horses. Experimental horses were selected from different working environments: (a) draft, agricultural works-n = 16, average age 8 years, (b) inconstant effort, leisure, n = 15, average age 7.5 years and (c) constant training, endurance, average age 3.5 years. Blood samples were collected in heparinized vials (50 UI/ml) before and after the workout. Alcoholic extracts (2 mu l/well, duplicate) of Taraxacum officinale (L) Weber, Symphytum officinale L., Equisetum palustre L., Viola tricolor L., Avena sativa L., Capsella bursa pastoris (L) Medik., Hypericum perforatum L., Chelidonium majus L. were investigated by blast transformation test and statistically interpreted (Students t test). In vitro responses were the highest to all tested extracts in endurance horses and the lowest in draft horses (p<0.01-0.001). C. bursa pastoris. was the most efficient in draft and endurance categories before the workout (draft 45.13 +/- 8.05, in leisure 48.09 +/- 24.13 and 53.44 +/- 9.32 in endurance horses), but not after the workout, where C. majus performed better (41.86 +/- 22.47 in leisure and 48.1 +/- 6.82 in endurance horses), probably due to its complex, protein-rich latex structure. The effects of plant extracts depended on the type of effort and the level of constant training rather than on the taxonomy of the plant

Skin Bacteriome and its Resistance to Antibiotics in Free Range Pigs

The cutaneous microbiome and also its resistance to antibiotics is exposed to change, depending on different habitat factors. This research investigates the composition of cutaneous microflora and its antibiotic resistance in pigs raised on free range farms (mix breed swine, from low input small farms). Swabs were collected from the skin surface and subjected to classical microbiological methods (simple broth and nutrient agar cultivation, colony isolation and biochemical API identification). The antibacterial resistance to gentamicin, streptomycin, oxitetracycline, tylosin, amoxacillin-clavulanic acid, marbofloxacin, tulatromycin, cefotaxime and doxycycline was estimated by Kirby Bauer method and multiple antibiotic resistance (MAR) index was calculated. Strains from Staphylococcus (sciuri and warnerii), Shigella spp., Kytococcus sedentarius, Salmonella spp. and Citrobacter freundii genera and species were identified in the collected samples. The most resistant was a S. warnerii strain, but the MAR index was high (0.33) in 50% of the strains. The most efficient antibiotic was cefotaxime and the least efficient was oxitetracycline. The results indicated the presence of antibiotic resistant ubiquitous and pathogenic strains in the investigated pigs which need caution, since they could express pathogenicity under appropriate conditions which low input farming system could provide

Bactericidal Capacity of Serum and Enhancement of Specific Cell-Mediated Immunity Subsequent to Hipopphae Rhamnoides Treatment in Pigs

Low-input farming subject’s swine to increased immune stress and subsequent infectious risk, which could be prevented by phytotherapy. Hypopphae rhamnoides is a widespread shrub in both Europe and Asia. The research aimed at investigating the influence of a whole fruit extract on antibacterial effect of the serum in extensively raised, two batches, sea buckthorn in vivo treated/untreated pigs. Scavenging effect over DPPH was used to estimate the antioxidant potential of the sea buckthorn commercial syrup (Steaua Divina©). Minimal inhibiting (MIC) and minimal bactericidal (MBC) capacity of the serum were tested and spectrophotometrically measured against Shigella spp., Kitococcus sedentarius, Staphylococcus werneri and Staphylococcus sciuri. The antioxidant activity of the syrup was intermediate (54.65 %). Sera from treated pigs had bactericidal and bacteriostatic effect on Shigella spp. and S. sciuri, respectively. Shigella spp. growth was inhibited at serum dilutions of ½-1/32 while much lower ½-1/4 dilutions were active against S. sciuri. In untreated pigs, the sera were inefficient in either stopping or totally inhibiting bacterial growth. The MBC and MIC values obtained for the sera of pigs treated with Hippophae rhamnoides syrup supported the positive effect of the in vivo treatment inducive of potential protection against bacterial diseases

Bidirectional Relationship Between Reduced Blood pH and Acute Pancreatitis: A Translational Study of Their Noxious Combination

Acute pancreatitis (AP) is often accompanied by alterations in the acid-base balance, but how blood pH influences the outcome of AP is largely unknown. We studied the association between blood pH and the outcome of AP with meta-analysis of clinical trials, and aimed to discover the causative relationship between blood pH and AP in animal models. PubMed, EMBASE, and Cochrane Controlled Trials Registry databases were searched from inception to January 2017. Human studies reporting systemic pH status and outcomes (mortality rate, severity scores, and length of hospital stay) of patient groups with AP were included in the analyses. We developed a new mouse model of chronic metabolic acidosis (MA) and induced mild or severe AP in the mice. Besides laboratory blood testing, the extent of pancreatic edema, necrosis, and leukocyte infiltration were assessed in tissue sections of the mice. Thirteen studies reported sufficient data in patient groups with AP (n = 2,311). Meta-analysis revealed markedly higher mortality, elevated severity scores, and longer hospital stay in AP patients with lower blood pH or base excess (P &lt; 0.001 for all studied outcomes). Meta-regression analysis showed significant negative correlation between blood pH and mortality in severe AP. In our mouse model, pre-existing MA deteriorated the pancreatic damage in mild and severe AP and, vice versa, severe AP further decreased the blood pH of mice with MA. In conclusion, MA worsens the outcome of AP, while severe AP augments the decrease of blood pH. The discovery of this vicious metabolic cycle opens up new therapeutic possibilities in AP

The Hypothermic Effect of Hydrogen Sulfide Is Mediated by the Transient Receptor Potential Ankyrin-1 Channel in Mice

Hydrogen sulfide (H2S) has been shown in previous studies to cause hypothermia and hypometabolism in mice, and its thermoregulatory effects were subsequently investigated. However, the molecular target through which H2S triggers its effects on deep body temperature has remained unknown. We investigated the thermoregulatory response to fast-(Na2S) and slow-releasing (GYY4137) H2S donors in C57BL/6 mice, and then tested whether their effects depend on the transient receptor potential ankyrin-1 (TRPA1) channel in Trpa1 knockout (Trpa1−/−) and wild-type (Trpa1+/+) mice. Intracerebroventricular administration of Na2S (0.5–1 mg/kg) caused hypothermia in C57BL/6 mice, which was mediated by cutaneous vasodilation and decreased thermogenesis. In contrast, intraperitoneal administration of Na2S (5 mg/kg) did not cause any thermoregulatory effect. Central administration of GYY4137 (3 mg/kg) also caused hypothermia and hypometabolism. The hypothermic response to both H2S donors was significantly (p &lt; 0.001) attenuated in Trpa1−/− mice compared to their Trpa1+/+ littermates. Trpa1 mRNA transcripts could be detected with RNAscope in hypothalamic and other brain neurons within the autonomic thermoeffector pathways. In conclusion, slow- and fast-releasing H2S donors induce hypothermia through hypometabolism and cutaneous vasodilation in mice that is mediated by TRPA1 channels located in the brain, presumably in hypothalamic neurons within the autonomic thermoeffector pathways

Synergistic antimicrobial potential of lactoferrin and oregano extract on potentially pathogenic ported bacteria isolated from swine

Key words: lactoferrin, oregano extract, antimicrobial potential, natural products, ported bacteria swine The widespread and uncontrolled use of antibiotics has led to the emergence of multi-drug resistant microorganisms. Thus, the use of substances with antimicrobial potential can represent reliable alternatives, thus contributing to the reduction of the load of multiresistant pathogens in the veterinary field. Lactoferrin is an iron binding glycoprotein present in exocrine secretions, with multiple biological functions, including antimicrobial potential. This study aimed to assess the antimicrobial capacity of lactoferrin alone and in combination with alcoholic extract of oregano (Origanum vulgare) on Staphylococcus spp. strains (n=5) isolated from nasal cavity of healty swine raised on low-input outdoor farms from North Western and Central Romania. The antibacterial activity was tested by the agar-well diffusion (Kirby–Bauer assay) and broth microdilution methods. The study findings confirmed the hypothesized enhanced antimicrobial properties of lactoferrin in combination with oregano extract against Staphylococcus spp. strains, compared to the two products tested alone and control antibiotic. Our results indicate a synergistic antimicrobial potential for two natural products thus may contribute to the improvement of animal welfare by reducing the load of potentially pathogenic, antibiotic-resistant bacteria. The work was supported by grant ERANET Core Organic Co-fund ROAM Free #249 ⁄ 2021, European Union’s Horizon 2020 research and innovation programme PPILOW project under grant agreement N°816172

THE POTENTIAL OF ALCOHOLIC PLANT EXTRACTS TO CONTROL PATHOBIONTS OF SWINE ON A LOW-INPUT FARM

Abstract Objective / Purpose: Plant extracts have long proven antimicrobial potential depending on both the plant and target animal species. Pathogenic bacteria expose animals to disease risk, thus their control being of utmost importance. This research aimed to investigate the natural potential of locally available traditional medicinal plants in controling the antibiotic resistante bacterial load in swine raised on low-input outdoor farms from North Western and Central Romania. Material and Methods: Aerobic bacterial strains (n=14) originating from the nasal cavities of extensively raised swine were subjected to biochemical identification (Vitek®2 Compact System) and further tested for susceptibility to antibiotics (n=12, antibiotic classes=6, Kirby-Bauer method). Simultaneously, the aromatogram technique was applied to indigenous Calendula officinalis, Saturaja hortensis, Coriandrum sativum, Artemisia absinthium, Cucurbita pepo, Allium sativum alcoholic extracts. Results: The antibiogram indicated a multiple antibiotic resistance (MAR) index > 0.2 in 86% of the bacteria (overall MAR=0.34). The highest average of inhibition diameters were observed with chloramphenicol (20.75±0.92 mm) and norfloxacin (20.68±1.55 mm), while the lowest was shown by cefotaxime (7.5±0.79 mm). Considering for intermediate susceptibility towards the tested antibiotics the threshold diameter of 13 mm, 12/14 strains were susceptible to C. pepo, 4/14 to C. sativum and 1/14 to C. officinalis extracts, while other extracts exerted insignificant antimicrobial activity. The average inhibition diameter was of 18.43±1.68 mm for C. pepo and of 11.11±0.68 mm for C. sativum extract. The lowest average was found in Allium sativum - 6.86±0.35 mm. Conclusion / Discussion: Some of the tested plant extracts could display a considerable antimicrobial activity on pathobionts of swine. These plants could enhance the welfare of the animals by reducing the potentially pathogenic, antibiotic resistante bacterial load, as an alternative to classical antibiotic therapy. Acknowledgements This research was funded by PPILOW project of the European Union’s Horizon 2020 programme, grant agreement 816172. Key Words: plant extracts, low-input swine farm, bacteria, antibiotic

Enhanced Bioactive Potential of Functionalized Injectable Platelet-Rich Plasma

Injectable platelet-rich fibrin (iPRF) is a frequently used platelet concentrate used for various medical purposes both in veterinary and human medicine due to the regenerative potential of hard and soft tissues, and also because of its antimicrobial effectiveness. This in vitro study was carried out to assess the cumulative antimicrobial and antibiofilm effect of iPRF functionalized with a multifunctional glycoprotein, human lactoferrin (Lf). Thus, the ability to potentiate cell proliferation was tested on keratinocytes and evaluated by the CCK8 test. The combinations of iPRF and Lf induced an increase in the proliferation rate after 24 h. The average cell viability of treated cultures (all nine variants) was 102.87% ± 1.00, and the growth tendency was maintained even at 48 h. The highest proliferation rate was observed in cultures treated with 7% iPRF in combination with 50 µg/mL of Lf, with an average viability of 102.40% ± 0.80. The antibacterial and antibiofilm activity of iPRF, of human lactoferrin and their combination were tested by agar-well diffusion (Kirby–Bauer assay), broth microdilution, and crystal violet assay against five reference bacterial strains. iPRF showed antimicrobial and antibiofilm potential, but with variations depending on the tested bacterial strain. The global analysis of the results indicates an increased antimicrobial potential at the highest concentration of Lf mixed with iPRF. The study findings confirmed the hypothesized enhanced bioactive properties of functionalized iPRF against both Gram-positive and Gram-negative biofilm-producing bacteria. These findings could be further applied, but additional studies are needed to evaluate the mechanisms that are involved in these specific bioactive properties

A COMPARATIVE EVALUATION OF PLANT EXTRACTS’ INHIBITING EFFECTES ON PORTED BACTERIA IN PIGS AND SMALL RUMINANTS

Abstract Continuously increasing antimicrobial resistance urges for research in identifying new active compounds agains bacterial pathogens. This research aimed to monitor the potential of traditional medicinal plants in efficient control of antibiotic resistant bacteriome in swine and small ruminants raised on low-input outdoor farms from North Western Romania. Aerobic bacterial strains (n=14) originating from the nasal cavities of extensively raised swine and sheep were subjected to biochemical identification (Vitek®2 Compact System) and further tested for susceptibility to antibiotics (n=12, antibiotic classes=6, Kirby-Bauer method). Simultaneously, the aromatogram technique was applied to alcoholic extracts Calendula officinalis, Saturaja hortensis, Coriandrum sativum, Artemisia absinthium, Cucurbita pepo, Allium sativum and essential oils of Anethum graveolens, Zingiber officinale, Geranium spp. and Lavandula angustifolia. The antibiogram indicated a multiple antibiotic resistance (MAR) index > 0.2 in 86% of the pigs and 33% in sheep (overall MAR=0.34 and 0.13, respectively). In pigs the highest average of inhibition diameters was observed with chloramphenicol (20.75±0.92 mm) and norfloxacin (20.68±1.55 mm), while the lowest was shown by cefotaxime (7.5±0.79 mm); in sheep the amikacin was the most efficient. Out of the plant extracts, C. pepo was the most effective in pigs (12 out of 14 strains) while the in sheep, the highest efficacy was recorded for the essential oil of Geranium spp. (26.87±8.2 mm) and the lowest activity was noted for the Anethum graveolens oil (15.04±2.2 mm). These plants could enhance the welfare of the animals by reducing the potentially pathogenic, antibiotic resistante bacterial load, as an alternative to classical antibiotic therapy. These results open the perspective of using essential oils in the prevention of staphylococcal infections. Key words: low input farms, sheep, pigs, plant extracts, antimicrobial resistanc