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

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

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

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

MICROFLORA PURTATĂ ȘI RISCUL ANTIBIOTICOREZISTENȚEI LA OVINE ÎN CREŞTEREA SEMI-INTENSIVĂ

Cea mai mare parte a florei bacteriene purtate se regăsește în tractul respirator și digestiv al animalelor sănătoase sau al oamenilor și nu exercită nici un efect patogen în circumstanțe fiziologice. Atunci când factorii favorizanți induc diminuarea imunității, tulpinile aparent inofensive pot deveni extrem de patogene pentru animale și consumatori sau oameni care lucrează în sectorul animal. Evaluarea încărcăturii microbiene de portaj la ovine crescute pe paşune în turme mixte cu caprine Identificarea populaţiilor bacteriene din mediul de viaţă a ovinelor Evaluarea rezistenţei la antibiotice a tulpinilor izolate de la ovine, cât şi a bacteriilor din mediu de viaţă a animalelor. Materiale și metode Scopul studiului a fost acela de a identifica și compara tulpinile bacteriene digestive și respiratorii portate de la oi (624) și capre (76) într-un efectiv mixt. Cercetările au fost efectuate pe 30 de animale sănătoase (15 capre și 15 oi) din același efectiv. Tampoanele nazale și rectale au fost cultivate pe bulion simplu timp de 24 de ore la 37°C și inseminare pe plăci de agar cu glucoză pentru încă 24 de ore la 37°C, inoculate și pe agar MacConkey și Chapman. Izolatele au fost identificate prin utilizarea sistemului de testare API 20 E pentru familia Enterobacteriaceae și API 20 Staph. pentru coloniile bacteriene crescute pe mediu Chapman. Testarea sensibilității la antibiotice s-a făcut prin metoda Kirby Bauer. Pentru interpretarea statistică a diferențelor de prevalență a fost utilizat testul exact Fisher (r pentru Windows). Rezultate și discuții Bacteriile identificate în turma mixta de oi şi capre demonstrează că examenul clinic şi diagnosticul de sănătate clinică nu sunt suficiente pentru eliminarea riscului patologic şi zoonotic, avându-se în vedere procentele relativ ridicate de bacterii condiţionat patogene. Există posibilitatea ca gradul mare de încărcare microbiană în saivanele în care sunt adăpostite oile şi caprele să fie datorat lipsei de igienă corespunzătoare, fiind impuse astfel verificări periodice ale parametrilor igienico-sanitari. O importanţă epidemiologică deosebită o au, indiferent de habitat, bacteriile cu potenţial patogen zoonotic cum sunt: Escherichia coli, izolată din toate arealele studiate, Serratia ficaria şi Serratia fonticola izolate de pe păşune. Unele specii bacteriene (Erwinia spp., Pantoea spp., Serratia ficaria), sunt bacterii fitopatogene, care prin acţiunea lor depreciază calitativ furajul animalelor. Notarea aprecierilor de susceptibil, intermediar, rezistent precum şi a variantelor intermediare a acestora (0,1,2,3 şi 4 ) Rezistenţa relativ ridicată la antibiotice din gama penicilinelor demonstrează expunerea îndelungată la acest antibiotic, a cărui eficienţa terapeutică este de multe ori variabilă Cele mai bune rezultate s-au obţinut la gentamicină şi doxiciclină cu toate acestea se recomandă o atenţie deosebită la alegerea unui antibiotic, indicaţia terapeutică fiind bazată pe rezultatele antibiogramei Rezultatele obţinute la testarea meticilinei pun în evidenţă tulpini de Staphylococcus xylosus şi Staphylococcus lentus emergente care se fac remarcate prin rezistenţă deosebită la acest antibiotic Concluzii Bacterii de origine fecală precum E. coli au fost identificate în sistemul respirator al caprelor, dar nu și al oilor, în timp ce tulpinile de E. cloacae au fost reprezentate în tractul respirator al oilor, dar nu și al caprelor. Deși turma a fost mixtă, au existat diferențe între izolatele bacteriene, ceea ce sugerează o susceptibilitate și un comportament de hrănire diferențiat la aceste specii. Cu toate acestea, izolatele bacteriene purtate de la capre și oi clinic sănătoase ar putea exercita efecte patogenice în circumstanțe stresante, subliniind importanța identificării precoce a agenților patogeni și a managementului sanitar precis al persoanelor auzite

Enhanced responses in antigen primed dogs after administration of a complex microbial immune stimulating compound

The research aimed to investigate the efficacy of antiparvoviral monovalent vaccine for puppies treated with a complex poly-bacterial compound. The experiment was carried out on 17 Labrador Retriever puppies, from 41 to 63 days of age. The two groups (experimental and control) were subjected to: a) a polybacterial preparation and antiparvoviral vaccine (n = 12) and b) to antiparvoviral vaccine (n = 5), respectively. One ml of a poly-bacterial preparation was administered on days 0, 2, 4, 6, while the antiparvoviral vaccine Duramune KF11 was injected on day 8 of the experiment. Phagocytic activity and total lysozyme concentrations were measured on days 0, 8 and 22 of the experiment. The global immunostimulating effects of the poly-bacterial preparation on the innate immune system of vaccinated puppies increased when compared to those of the control variant

IS BOVINE MASTITIC BACTERIOME SENSITIVE TO PLANT EXTRACTS?

Abstract One of the most impacting diseases of dairy cows worldwide, due to the difficulties to diagnose subclinical development and to treat the clinical disease is represented by mastitis. In this study the several plant extracts and essential oils were assessed for antimicrobial efficacy on bacteria isolated from subclinical cases of mastitis. Milk samples were obtained from Romanian Spotted extensively raised dairy cows (n=20), cohabiting on the same farm with sheep and pigs, which were diagnosed with subclinical mastitis. The microbiome components were identified by use of classical bacteriological methods and cultivated against Melissa officinalis and propolis alcoholic extracts and essential oils of Rosmarinus officinalis, Thymus vulgaris and Lavandula angustifolia in the Kirby-Bauer well diffusion method. S. sciuri, Staphylococcus spp, E. coli, Enterococcus faecium were isolated from the milk samples, showing a high MAR index, thus eliminating the possibility of therapy with 12 of the 14 antibiotics tested in some isolates. The best antibacterial effect was shown by Thymus vulgaris essential oil (25±5.6 mm inhibition) versus the weakest effect of Lavandula angustifolia essential oil (17.5±8.2mm inhibition). Staphylococcus spp. showed the highest sensitivity, while E.coli strains were much more resistant. When compared to the essential oil the Melissa tincture was less effective (11.3±3.6mm versus 12.3±4.3mm) but comparable to amoxicillin, amoxicillin/clavulanic acid and stronger than cefoperazone. The propolis extracts used to control bacterial growth have shown in vitro efficacy, the effect depending on the concentration used. Both tincture and essential oils proved to be efficient depending more on the strain than on the solvent type. These results are opening the prospect of using plant extracts, essential oils and propolis as an alternative to antibiotics in the treatment of mastitis, leading to safer products, with less antibiotic residues in milk. Keywords: subclinical mastitis, microbiome, antibiotic resistance, medicinal plants, dairy cow

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