Evaluation of the in vitro efficacy of Lactobacillus perolens CRL 1724 as a potential probiotic strain for the prevention of bovine mastitis

IGNACIO FROLA; MATIAS PELLEGRINO; JOSE GIRAUDO; E, DUCROS; MARIA ELENA FÁTIMA NADER; CRISTINA BOGNI

Santiago de Chile

Conferencia; XXVI World Buiatrics Congress; 2010

                    Evaluation of the in vitro efficacy of Lactobacillus perolens CRL 1724, as a potential probiotic strain for the prevention of bovine mastitis I. Frola1, M. Pellegrino1, J. Giraudo2, E. Ducros1, M.E.F. Nader3 and C. Bogni1. 1Dpto. Microbiología e Inmunología, Fac. Cs. Ex. Fco-Qcas y Naturales, 2Dpto. Patología Animal, Fac. Agronomía y Veterinaria, Universidad Nacional de Río Cuarto, Ruta 36 Km 601, 5800 Río Cuarto, Córdoba, Argentina. 3CERELA-CONICET Centro de Referencia para lactobacilos, Chacabuco 145, 400 Tucumán.   Bovine mastitis, defined as an inflammation of the mammary gland, is one of the most expensive diseases in dairy farming. Our research group is involved in the design of veterinary probiotic products to be applied intramammary in dairy cows to prevent mastitis. In a previous work, a strain of Lactobacillus perolens was isolated from milk of healthy dairy cows and deeply characterized. This strain was considered as potentially probiotic because of the high hydrophobicity, moderate autoaggregation, organic acid production and inhibition of S. dysgalactiae and S. uberis. In the present work, we investigate the role of L. perolens CRL 1724 for inhibiting and co-aggregate with potential pathogens causing bovine mastitis, as well as the in vitro capacity of this strain to adhere to bovine teat canal epithelial cells (BTCC). For inhibitory activity, the cross striations technique in MRS 1.2% agar was used. L. perolens was faced to fourteen potential pathogen bacteria characterized as Staphylococcus spp. (2), Streptococcus spp. (7) Enterococcus spp. (2), Klebsiella spp. (1), E. coli (1) and Pseudomonas spp (1). For co-aggregation assay 500µl of L. perolens CRL 1724 (109cfu/ml) were mixed with 500µl of each potential pathogen bacteria (14) by vortexing, incubated at 37°C and the Gram-stained suspensions obtained evaluated though optical microscope. For adherence assay, 500 µl of BTCC (obtained by scrapping the teat canal wall and their viability determined bytrypan blue-stained exclusion) anda suspension of 500 µl L. perolens CRL 1724 (idea de la cantidad de UFC????) were mixed and incubated under low agitation conditions at 37ºC for 1h. The tubes were centrifuged, the supernatant discarded and the pellets  suspended in MEM, and washed under the same conditions. Bacterial binding to BTCC were examined by optical microscope (Gram and Hematoxylin-Eosin stains) and fixed with 3.16% glutaraldehyde in 0.1M pH 7.4 phosphate buffer to be examined under Scanning Electron Microscope. L. perolens CRL 1724 presented a high percentage (71.4%) of inhibition zone against all The potential pathogen bacteria assayed. L. perolens CRL 1724 was able to co-aggregate most of the potential pathogen bacteria, but a low co-agregation was observed with Pseudomonas spp. and Klebsiella spp. A high pattern of adhesion of L. perolens CRL 1724 to BTCC (14.4 and 75% for the Adhesion index and Percentage of adhesion respectively) was observed. L. perolens CRL 1724 BTCC adhesion was confirmed by Scanning Electron Microscopy.  The morphologic or ultrastructural modifications of the cells of the cells in contact of with lactobacilli adheres were not observed. The results obtained in this work provide the main and first evidence to considered L. perolens CRL 1724 as a potential probiotic strain to be included in the generation of non-antibiotic formulation for the treatment and prevention of mastitis in dairy cows.