Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy HAF © 2013 Vol. V, No. 1s ISSN: 2283-3927 l Water buffaloes mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yield. The emergence of antibiotic resistance has led to investigate strategies in order to avoid or minimize the antibiotic use, especially during subclinical mastitis (SM) (Moroni et al., 2006). Lactobacillus rhamnosus is part of the normal gut microflora, having meanwhile an immunostimulatory activity (Fong et al., 2016). The aim of this study was to investigate the change of milk microbiota after the therapeutic treatment of mammary gland quarters affected by subclinical mastitis with inactivated cultures of Lactobacillus rhamnosus and antibiotics. A number of 43 quarters from 20 pluriparous animals and affected by subclinical mastitis (SM), with no signs of clinical mastitis and aerobic culture positive for udder pathogens were included in the study; milk samples were positive for coagulase-negative or positive Staphylococci and/or Streptococcus agalactiae. A total of 11 quarters were locally treated with antibiotic (amoxicillin trihydrate), 15 with Lactobacillus rhamnosus and 17 with PBS as negative control, by means of intramammary injection. Samples were collected at two time points, T0 (pre-treatment) and T5 (after 5 days post-treatment) and V4 region of 16S rRNA gene was amplified by PCR and sequenced using Ion Torrent Personal Genome Machine. The software Quantitative Insights Into Microbial Ecology (QIIME version 2) was used to analyse data. Microbiota composition was evaluated in terms of taxonomy at phylum, family and genus level. Microbiota structure was investigated through alpha and beta diversity analysis which take into account differences within and among samples, respectively. Non-parametric test, namely Wilcoxon signed rank and Kruskal-wallis followed by Dunn test, were used to perform statistical analysis for paired and unpaired groups, respectively. Keywords Water buffalo, milk microbiota, subclinical mastitis. CORRESPONDING AUTHOR Carlotta Catozzi carlotta.catozzi@unimi.it JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Effect of inactivated cultures of Lactobacillus rhamnosus and antibiotics on subclinical mastitis quarter milk microbiota. C. Catozzi*,1 , A. Cuscó Martí2, C. Lecchi1, V. Zamarian1, J. Viñes Pujol2, S. D'Andreano2, A. Martuccello3, G. Cappelli3, C. Grassi3, C. Marianelli4, L. D’Angelo3, E. De Carlo3, D. Vecchio3, A. Sanchez Bonastre5, O. Francino5, F. Ceciliani1. 1 Department of Veterinary Medicine, Università degli Studi di Milano, Via Celoria 10, Milano, Italy. 2 Vetgenomics. Ed Eureka. PRUAB. Campus UAB, Bellaterra, Barcelona, Spain. 3 Istituto Zooprofilattico Sperimentale del Mezzogiorno, National Reference Centre for Hygiene and Technologies of Water Buffalo Farming and Productions, Via delle Calabrie, Salerno, Italy. 4 Unit of Prophylaxis and Control of Bacterial Zoonoses. Department Of Veterinary Public Health and Food Safety. Istituto Superiore di Sanità. Viale Regina Elena 299, Rome, Italy. 5 Molecular Genetics Veterinary Service (SVGM), Veterinary School, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy 34 HAF © 2013 Vol. V, No. 1s ISSN: 2283-3927 Regarding taxonomy, the microbiota composition of SM quarters showed no major changes after PBS treatment, while differed after antibiotic treatment where Staphylococcus decreased its relative abundance from 41% at T0 to 3% at T5. Lactobacillus rhamnosus induced a less dramatic change in milk microbiota, although the relative abundance of some genera was found to be modified, among which an increase of Pseudomonas from 1.5% at T0 up to 4% at T5. The taxonomy at genus level is shown in Figure 1. However, beta diversity showed no differences between the microbiota structure of quarters treated with Lactobacillus rhamnosus and PBS (T0 vs T5). The effect of the treatment was different between antibiotic- vs PBS- treated groups and antibiotic- vs Lactobacillus rhamnosus- treated groups. In conclusion, this study allowed to characterize the microbiota in milk from animals treated with Lactobacillus rhamnosus and antibiotics; while changes in milk microbiota after antibiotic treatment were evident, changes after Lactobacillus rhamnosus were more limited. Following investigation will include the study of the microbiota changes of antibiotic-treated quarters during more than two time points, in order to investigate the colonization of mammary gland after antibiotic treatment during a time course. References Moroni, P., Sgoifo Rossi, C., Pisoni G., Bronzo V., Castiglioni, B., Boettcher PJ. 2006 Relationships between somatic cell count and intramammary infection in buffaloes. J. Dairy Sci. 89, 998–1003. Fong, FL., Shah, NP., Kirjavainen, P., El-Nezami, H,. 2016 Mechanism of Action of Probiotic Bacteria on Intestinal and Systemic Immunities and Antigen-Presenting Cells. Int Rev Immunol. 35(3):179-88. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% SM_PBS_T0 SM_PBS_T5 SM_L_T0 SM_L_T5 SM_A_T0 SM_A_T5 Low abunda nce taxa Pseudomonadaceae Pseudomonadaceae;g __Pseudom onas Mor axella ceae;g__Psychrobacter Mor axella ceae;g__A cinetobacter Halomona da ceae;g__Halomonas Enteroba cteriaceae Enteroba cteriaceae;g__Escherichia Idiomari na ceae Rhodocyclaceae;g __Hydrogenophilus Comamonadacea e Comamonadacea e;g__Delftia Sphing om onadaceae;g__Sphing om onas Xanthobacteracea e Methylobacteria ceae;g__Methylobacter ium Methylobacteria ceae Bradyrhizobiaceae Bradyrhizobiaceae;g__Bra dyrhizobium Ruminococcaceae Peptostreptococca ceae Lachnospir aceae Clostridiaceae [Mogi ba cteriaceae] Streptococca ceae;g__S treptococcus Carnoba cteria ceae;g__Granulicatella Aerococcacea e Aerococcacea e;g__Facklam ia Aerococcacea e;g__Alkali ba cterium Staphylococcaceae;g__Staphylococcus Staphylococcaceae;g__Sali nicoccus Staphylococcaceae;g__Jeotga licoccus Planococcacea e;g __Soliba cillus Planococcacea e;g __Ly sinibacillus Bacilla ceae;g__Natronoba cillus Xenococcacea e Fla vobacteriacea e [Weeksellaceae];g__Chryseobacterium Cytophag acea e;g __Hym enobacter Por phyromonadacea e Bacteroidaceae;g__5-7N15 Bacteroidaceae [Pa raprevotellaceae];g__CF231 Propionibacteria ceae;g__Propionibacterium Noca rdioidaceae Noca rdiacea e;g __Rhodococcus Micrococcacea e Micrococcacea e;g __Nesterenkonia Microbacteriaceae Intraspor angiaceae Dietziacea e;g__Dietzia Corynebacteri aceae;g __Coryneba cterium Actinomycetaceae Deinococcacea e;g__Deinococcus Figure 1: Microbiota composition at genus level (relative abundance > 1%). SM_PBS_T0: subclinical quarters treated with PBS (pre-treatment) SM_PBS_T5: subclinical quarters treated with PBS (post-treatment) SM_L_T0: subclinical quarters treated with Lactobacillus rhamnosus (pre-treatment) SM_L_T0: subclinical quarters treated with Lactobacillus rhamnosus (post-treatment) SM_A_T0: subclinical quarters treated with antibiotic (pre-treatment) SM_A_T5: subclinical quarters treated with antibiotic (post-treatment) http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en