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Evaluation of beneficial Lactic Acid Bacteria strains as a potential probiotic for the prevention of bovine mastitis

Ignacio D. Frola1, Matías S. Pellegrino1, José Á. Giraudo2, María Elena F. Nader-Macias3 and Cristina I. Bogni1*
  • 1 Universidad Nacional de Río Cuarto, Department of Microbiology and Immunology, Faculty of Cs. Ex. Fco-Qcas y Naturales, Argentina
  • 2 Universidad Nacional de Río Cuarto, Department of Patology, Faculty of Veterinary, Argentina
  • 3 CERELA-CONICET (Centro de Referencia para Lactobacilos-Consejo Nacional de Investigaciones Cientifícas y Técnicas de Argentina)., Department of Preventive Microbiology, Argentina

The conventional methods for the control of mastitis are basically supported by the adoption of preventive strategies including diagnosis, segregation of the animals, use of improved hygiene and therapeutic protocols, with antibiotics frequently applied. Antibiotics used routinely in dry cow therapies are questioned by the adverse effects in the human health (Barkema et al. 2006; McDougall et al. 2009). Probiotics, live microorganisms administered in adequate amounts conferring a health benefit on the host, constitute an alternative tool to bovine mastitis treatment (FAO & WHO, 2008). Probiotics may exert their beneficial effects on the host health by several mechanisms: adhesion to epithelial cells, colonization, biofilm formation, production of biosurfactants, aggregation and co-aggregation, production of antagonistic metabolites (organic acids, hydrogen peroxide, bacteriocins), competition for nutrients, production of enzymes and/or immunomodulation (Espeche et al. 2009). Lactic acid bacteria (LAB) are the main components of the indigenous microbiota of the teat canal and therefore are optimal candidates to design a specie-specific probiotic product to prevent mastitis (Espeche et al. 2009; Giannino et al. 2009). In the present study, the preventive effect of LAB selected by their beneficial properties: Lactobacillus perolens CRL 1724, Enterococcus hirae CRL 1835 and Lactococcus lactis subsp. lactis CRL 1655, after their infusion by the intramammary route, was investigated. The levels of milk specific antibodies, neutrophil phagocytosis, milk appearance, somatic cell counts (SCC) and LAB recovery in milk were determined, and also the clinical signs of the udder. The LAB used in this study were isolated from milk of Holstein cows from Córdoba and Tucumán, Argentina, and genetically identified by 16S rRNA gene sequencing by Espeche et al. (2009). The strains were selected as potentially probiotic bacteria because of their hydrophobicity index, auto-aggregation and ability to produce antagonistic metabolites (Espeche et al. 2009, 2012, Frola et al. 2012, 2013). Before to develop the intramammary formulation (IMF) the following assays were done: a) antimicrobial activity of LAB against 15 mastitis-causing pathogens (MCPs) (Str. agalactiae ATCC27956, Str. dysgalactiae ATCC27957, Str. uberis 102, Str. uberis ATCC27958, Staph. hyicus 112249, Str. bovis ATCC27960, Enterococcus (E.) faecalis 19433, E. faecium 35667, Escherichia coli ATCC35218, Klebsiella (K.) pneumoniae ATCC10031, Staph. epidermidis ATCC14990, Staph. aureus ATCC25923, Staph. aureus RC108, Escherichia coli 345 and Pseudomonas spp.) by the streak line method (Hütt et al. 2006) b) agregation between LAB strains and MCPs by mixing 109 cfu/ml of LAB with 108 cfu/ml of each MCP during 4 h at 37 ºC in shaker and Gram-stained observation, c) adhesion of LAB strains to bovine teat canal epithelial cells (BTCEC) was done by scraping the teat canal wall of udders with a Medibrush XL (Medical Engineering Co.) and suspended immediately in 1 ml of Eagle’s minimum essential media (MEM; Gibco; pH 7.0). Equal volumes (500 µl) of BTCEC and LAB suspensions were mixed and incubated under low agitation 1 h at 37 ºC. Bacterial binding to BTCEC were examined by Gram stain and results expressed as Percentage of Adhesion (PA) and Adhesion Index (AI), and d) auto-inhibition activity between LAB by the streak line method. The animal trial was performed in a dairy farm in Córdoba, Argentina. Twenty seven non-lactating Holstein cows with different degree of udder health were IM infused with LAB suspension (6 x106 cfu/ml in saline solution prepared from 2 x 106 cfu/ml of each LAB). Healthy udders were considered as free of MCP and SCC < 2 x 10 5 cell/ml in milk and without clinical signs. The animals were IM infused in the four quarters after the evening milking and separated into 3 groups: Group A (GA), where 10 cows were infused with 1 ml of the LAB bacterial suspension, Group B (GB) where 10 cows were infused with a commercial antibiotic (Bovigam, Bayer) and Group C (GC) included 7 animals without treatment used as control (Figure 2). Samples were obtained 30 and 10 days before the LAB inoculation (D-30, D-10), immediately before inoculation (D0) and 60 days (D60) post inoculation of IMF. The levels of specific antibodies in milk were determined by enzyme-linked inmunosorbent assay (ELISA). To investigate milk serum capacity of inoculated cows to increase neutrophil phagocytosis, neutrophils were isolated from healthy cows and incubated with FITC labeled L. perolens and L. lactis opsonised with pooled milk sera of GA animals. Quantitative phagocytosis was determined under oil-immersion fluorescence microscopy (1000X) and phagocytosis index (PI) calculated. Milk or dry secretion samples collected from each quarter were plated on TSA + 5% sheep blood, for detection of MCP by standard biochemical tests or MRS pH….(Man Rogosa Sharpe, Britania) agar plates for LAB isolation. Lact. perolens CRL 1724, Enter. hirae CRL 1835 and Lact. lactis subsp. lactis CRL 1655 were Gram stained for bacterial morphology, and catalase activity, nitrate reduction, indole production and streptomycin resistance were determined. Milk SCCs were quantified by Somacount 300 (Bentley) and the udder clinical signs recorded. Animal´s care was achieved in accordance with The International Guiding Principles for Biomedical Research Involving Animals. The LABs selected to be included in the potential probiotic formulation, Lact. perolens CRL 1724, Enter. hirae CRL 1835 and Lact. lactis subsp. lactis CRL 1655, showed 80%, 80% and 73 % of inhibition of the 15 MCP, respectively. Enter. hirae CRL 1835, and Lact. lactis subsp. lactis CRL 1655 evidenced the highest inhibitory effect against Str. dysgalactiae ATCC27957, producing a complete inhibition. Lactob. perolens CRL1724 and Enter. hirae CRL1835 showed 100% of co-aggregation with all of the MCP assayed, whereas Lactoc. lactis subsp. lactis CRL 1655 produced 93%, but without co-aggregation with Pseudomonas spp. When adherence to BTCEC was quantified, AI and PA were 14.4, 27.9, 36.7 and 75%, 73% and 96 % for Lactob. perolens CRL 1724, Lactoc. lactis subsp. lactis CRL 1655 and Enteroc. hirae CRL 1835, respectively. None of the BAL showed ability to self-inhibition by co-culture. Referred to animal trials, no clinical signs or teat damage were observed after IMF infuses of 6 x 106cfu/ml in non-lactating cows during the dry period. A significant increase in specific IgG was observed (p < 0.05) in animals of

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References

Barkema, H.W., Schukken, Y.H., Zadoks, R.N., 2006. Invited review: The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. Journal of Dairy Science 89, 1877-1895.
Espeche, M.C., Otero, M.C., Sesma, F., Nader-Macias, M.E.F., 2009. Screening of surface properties and antagonistic substances production by lactic acid bacterial isolated from the mammary gland of healthy and mastitis cows. Veterinary Microbiology 135, 346-357.
Espeche, M.C., Pellegrino, M., Frola, I., Larriestra, A., Bogni, C, Nader-Macias, M.E.F., 2012. Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis. Anaerobe 18 (1), 103-109.
FAO, WHO, 2008. Health and nutritional properties of probiotics in food, including powder milk with live lactic acid bacteria. http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf. (Accessed 22 November 2014).
Frola, I.D., Pellegrino, M.S., Espeche, M.C., Giraudo, J.A., Nader-Macias, M.E., Bogni, C.I. 2012. Effects of intramammary inoculation of Lactobacillus perolens CRL1724 in lactating cows' udders. Journal of Dairy Reserch 79 (1), 84-92.
Frola, I.D., Pellegrino, M.S., Magnano, G., Giraudo, J.A., Espeche, M.C., Nader-Macias, M.E., Bogni, C.I. 2013. Histological examination of non-lactating bovine udders inoculated with Lactobacillus perolens CRL 1724. Journal of Dairy Reserch 80 (1), 28-35.
Giannino, M.L., Aliprandi, M., Feligini, M., Vanoni, L., Brasca, M., Fracchetti, F., 2009. A DNA array based assay for the characterization of microbial community in raw milk. Journal of Microbiological Methods 78, 181-188.
Hütt, P., Shchepetova, J., Lõivukene, K., Mikelsaar, M., 2006. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. Journal of Applied Microbiology 100, 1324-1332.
McDougall, S., Parker, K.I., Heuer, C., Compton, C.W.R., 2009. A review of prevention and control of heifer mastitis via non-antibiotic strategies. Veterinary Microbiology 154, 177-185.

Keywords: Bovine Mastitis, Probiotics, Staphylococcal Infections, Preventive Medicine, Inmune response

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Poster Presentation

Topic: Veterinary and Comparative Immunology

Citation: Frola ID, Pellegrino MS, Giraudo JÁ, Nader-Macias MF and Bogni CI (2015). Evaluation of beneficial Lactic Acid Bacteria strains as a potential probiotic for the prevention of bovine mastitis. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00231

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Received: 17 Apr 2015; Published Online: 14 Sep 2015.

* Correspondence: Dr. Cristina I Bogni, Universidad Nacional de Río Cuarto, Department of Microbiology and Immunology, Faculty of Cs. Ex. Fco-Qcas y Naturales, Río Cuarto,Córdoba, 5800, Argentina, cbogni@exa.unrc.edu.ar