Supplementary online material of Beneficial Microbes DOI: http://dx.doi.org/10.3920/bm2015.0048. Bovine mastitis may be associated with the deprivation of gut Lactobacillus C. Ma, J. Zhao, X. Xi, J. Ding, H. Wang, H. Zhang and L.Y. Kwok* Table S1. Somatic cell count (SCC) of milk samples. Health and subclinical mastitis group Mastitis group Cow number SCC ( 10 3 /ml) Cow number SCC ( 10 3 /ml) 1 18 37 1,049 2 25 38 1,087 3 29 39 1,102 4 31 40 1,225 5 31 41 1,399 6 33 42 1,525 7 33 43 1,756 8 36 44 1,759 9 37 45 2,008 10 40 46 2,070 11 41 47 2,071 12 46 48 2,117 13 50 49 2,338 14 55 50 2,710 15 73 51 3,104 16 75 52 3,432 17 80 53 3,690 18 82 54 3,910 19 89 55 4,091 20 91 56 4,132 21 91 57 4,851 22 94 58 5,419 23 97 59 5,565 24 116 60 5,630 25 131 61 5,664 26 137 62 5,801 27 151 63 5,965 28 154 64 6,282 29 174 65 6,797 30 200 66 7,435 31 215 67 8,817 32 226 68 9,094 33 250 69 9,319 34 258 70 10,189 35 273 71 11,534 36 275 72 11,698 1
Table S2. Oligonucleotide primers used in this study. Target microbes DNA Sequence (5-3 ) Total bacteria Fungi Bifidobacterium Bacteroides Enterococcus Staphylococcus Streptococcus E.coli Lactococcus lactis Lactobacillus L. helveticus L. salivarius L. gasseri CGGCAACGAGCGCAAC CC CCATTGTAGCACGTGTG TAGCC GAGGAAGTAAAAGTCG TAACAAGGTTTC CAAATTCACAAAGGGT AGGATGATT GCGTGCTTAACACATGC AAGTC CACCCGTTTCCAGGAG CTATT GAGAGGAAGGTCCCCC AC CGCTACTTGGCTGGTTC AG CCTTATTGTTAGTTGCC ATCATT ACTCGTTGTACTTCCCA TTGT GGCCGTGTTGAACGTG GTCAAATCA TIACCATTTCAGTACCTT CTGGTAA GAAGAATTGCTTGAATT GGTTGAA GGACGGTAGTTGTTGA AGAATGG CATGCCGCGTGTATGAA GAA CGGGTAACGTCAATGA GCAAA AGCAGTAGGGAATCTTC GGCA GGGTAGTTACCGTCACT TGATGAG AGCAGTAGGGAATCTTC CA CACCGCTACACATGGA G CTCTTCTCGGTCGCCTT G GAAGTGATGGAGAGTA GAGATTA TAAACAAAGTATTCGAT AAATGTACAGGTT GTCGTAACAAGGTAGC CGTAGGA TGCTATCGCTTCAAGTG CTT AGCGACCGAGAAGAGA Target gene 18s tuf tuf -23s -23s -23s -23s -23s Standard strain Escherichia coli ATCC 11775 Saccharomyces DSM 70412 Bifidobacterium ATCC 15700 bayanus breve Bacteroides fragilis ATCC 25285 Enterococcus canis DSM- 17029 Staphylococcus ATCC 12600 aureus Streptococcus salivarius subsp. thermophilus DSM 20617 Escherichia coli ATCC 11775 Lactococcus lactis subsp. hordniae DSM-20450 L. helveticus CGMCC 1.1877 L. helveticus CGMCC 1.1877 Reference (Denman & McSweeney, (Denman & McSweeney, (Zwielehner et al., 2009) (Zwielehner et al., 2009) (Collado et al., 2009) (Collado et al., 2009) (Collado et al., 2009) (Furet et al., 2009) L. salivarius DSM 20555 L. gasseri ATCC33323 2
L. ruminis L. buchneri L. sake L. acidophilus L. casei L. delbrueckii L. paracasei L. plantarum L. reuteri GAGA CACCGAATGCTTGCAYT CACC GCCGCGGGTCCATCCA AAA AGATTACTGACGCATTG GTTA GGACCAATGGAGCAAC TGAA GATAAGCGTGAGGTCG ATGGTT GAGCTAATCCCCCATAA TGAAACTAT GAAAGAGCCCAAACCA AGTGATT CTTCCCAGATAATTCAA CTATCGCTTA CTATAAGTAAGCTTTGA TCCGGAGATTT CTTCCTGCGGGTACTGA GATGT CACTTGTACGTTGAAAA CTGAATATCTTAA CGAACTCTCTCGGTCGC TTT ACATCAGTGTATTGCTT GTCAGTGAATAC CCTGCGGGTACTGAGAT GTTTC TGGATCACCTCCTTTCT AAGGAAT TGTTCTCGGTTTCATTAT GAAAAAATA ACCGAGAACACCGCGT TATTT CATAACTTAACCTAAAC AATCAAAGATTGTCT -23s -23s -23s -23s -23s -23s -23s -23s -23s L. ruminis DSM 20403 L.buchneri DSM 20057 L. sakei subsp. sakei DSM 20017 L. acidophilus DSM 20079 (Haarman & Knol, L. casei DSM 20011 (Haarman & Knol, L. delbrueckii subsp.indicus DSM 15996 L. paracasei subsp. paracasei DSM 5622 L. plantarum subsp. Argentoratensis DSM 16365 (Haarman & Knol, (Haarman & Knol, (Haarman & Knol, L. reuteri DSM 20016 (Haarman & Knol, 3
Table S3. Differences in bacterial quantities in the faecal and milk samples of healthy and mastitis cows. Microbial groups Milk Low SCC group Mean Median, Total bacteria 8.00±0.13 7.78, 6.69-10.35 Fungi 2.93±0.05 3.01, 2.35-3.30 Bacteroides 4.13±0.03 4.09, 3.70-4.53 E. coli 4.12±0.11 4.00, 3.16-6.34 Enterococcus 3.85±0.08 3.78, 3.13-5.26 Staphylococcus 5.03±0.12 4.93, 3.77-6.66 Streptococcus 5.77±0.08 5.85, 4.69-6.64 Bifidobacterium 3.46±0.07 3.40, 2.67-4.66 Lactobacillus 6.34±0.11 6.11, 5.28-7.97 Lactococcus 4.12±0.21 4.30, 2.14- lactis 6.16 L. salivarius 2.31±0.03 2.29, 2.04-2.96 L. sake 2.43±0.04 2.38, 2.15-3.42 L. ruminus 2.73±0.05 2.66, 2.30-3.75 L. reuteri 3.95±0.09 4.15, 2.07-4.24 L. plantarum 2.76±0.07 2.91, 1.95-3.62 L. paracasei 3.38±0.05 3.51, 2.73-3.90 L. helveticus 3.90±0.17 4.48, 1.90-4.77 High SCC group Mean Median, 8.77±0.15 9.02, 7.36-10.5 2.63±0.06 2.67, 1.73-3.11 3.72±0.04 3.74, 3.00-4.08 5.11±0.12 4.85, 4.30-7.14 4.92±0.13 4.90, 3.85-6.55 5.29±0.11 5.22, 4.38-6.63 6.04±0.1 6.22, 4.73-7.37 3.00±0.08 2.88, 2.40-4.30 5.97±0.08 5.97, 5.18-6.92 2.51±0.20 2.16, 1.14-6.84 1.90±0.04 1.89, 1.30-2.79 2.11±0.05 2.12, 1.18-2.62 2.34±0.04 2.30, 1.96-3.07 1.99±0.04 1.98, 1.41-2.65 1.88±0.06 1.88, 1.21-2.97 2.51±0.05 2.50, 1.87-3.21 1.97±0.07 1.95, 1.24-3.99 Faeces Response Low SCC group P-value of high Mean Median, SCC group 0.00043 11.63±0.10 11.71, 10.40-13.09 0.00021 6.04±0.06 6.04, 5.25-7.35 0.00000 9.23±0.08 9.17, 8.26-10.27 0.00000 7.15±0.08 7.17, 6.06-7.95 0.00000 7.47±0.07 7.38, 6.87-8.73 0.12980-6.46±0.06 6.43, 5.21-7.14 0.01007 7.05±0.13 7.39, 5.14-7.87 0.00002 6.92±0.08 6.88, 6.01-7.81 0.03471 8.04±0.08 8.09, 7.03-8.78 0.00000 5.02±0.08 5.00, 3.91-6.88 0.00000 5.29±0.07 5.30, 4.46-6.04 0.00000 4.62±0.05 4.59, 3.97-5.16 0.00000 4.74±0.05 4.74, 4.27-5.5 0.00000 4.90±0.07 4.95, 3.75-5.55 0.00000 4.82±0.07 4.81, 4.05-5.92 0.00000 4.46±0.09 4.48, 3.31-5.52 0.00000 5.00±0.05 5.00, 4.44-5.76 High SCC group Mean Median, 11.84±0.09 11.85, 10.69-12.80 6.05±0.07 6.05, 5.19-7.07 9.25±0.08 9.11, 8.56-10.36 6.93±0.07 6.87, 5.98-7.89 7.85±0.08 7.83, 7.00-8.82 6.54±0.08 6.53, 5.28-7.38 8.01±0.13 8.12, 6.17-8.90 6.90±0.09 6.68, 5.95-8.26 7.66±0.08 7.59, 6.36-8.88 5.09±0.08 5.06, 4.63-6.98 4.90±0.05 4.82, 4.42-5.68 4.13±0.08 4.15, 2.75-5.22 4.60±0.04 4.60, 4.22-5.08 4.87±0.05 4.84, 4.20-5.68 4.71±0.06 4.67, 4.22-5.74 4.43±0.06 4.42, 3.66-5.52 4.93±0.04 4.92, 4.18-5.43 Response P-value of high SCC group 0.11620-0.67280-0.96860-0.04322 0.00053 0.39520-0.00000 0.73970-0.00240 0.71430-0.00012 0.00000 0.03012 0.53190-0.14470-0.76540-0.25290-4
L. gasseri 3.65±0.04 3.62, 3.28-4.67 L. delbrueckii 2.90±0.05 2.96, 1.94-3.39 L. casei 3.76±0.10 3.77, 2.68-5.12 L. buchneri 3.41±0.04 3.37, 2.98-3.86 L. acidophilus 4.08±0.05 4.05, 3.56-5.08 3.72±0.04 3.72, 3.28-4.92 1.68±0.07 1.63, 0.86-2.83 3.19±0.09 3.08, 2.22-4.50 3.12±0.06 3.09, 2.45-4.39 3.84±0.05 3.76, 3.32-4.52 0.17120-4.73±0.06 4.73, 4.21-5.96 0.00000 4.77±0.05 4.77, 4.24-5.35 0.00017 5.52±0.09 5.43, 4.53-6.65 0.00000 4.42±0.05 4.39, 3.82-4.98 0.00071 5.45±0.05 5.45, 4.76-6.21 4.65±0.03 4.65, 4.14-5.05 4.63±0.03 4.63, 4.05-4.98 5.56±0.07 5.44, 4.85-6.60 2.78±0.16 2.48, 1.76-4.68 5.14±0.06 5.14, 4.02-5.79,, and - indicate increase, decrease, and no significant difference comparing bacterial quantity of low to high SCC group. Data are expressed in Log gene copy number per gram/ml of sample. 0.21730-0.00973 0.61630-0.00000 0.00012 5
Table S4. Spearman correlation between individual faecal Lactobacillus species and somatic cell count. Lactobacillus r P-value species L. buchneri -0.5449 0.0000 L. salivarius -0.3697 0.0014 L. casei 0.1248 0.2964 L. sake -0.4859 0.0000 L. gasseri -0.0612 0.6096 L. ruminis -0.2698 0.0219 L. reuteri -0.0465 0.6979 L. helveticus -0.1626 0.1724 L. acidophilus -0.4118 0.0003 L. paracasei 0.0195 0.8706 L. delbrueckii -0.2809 0.0168 L. plantarum -0.2025 0.0881 References Collado, M.C., Delgado, S., Maldonado, A. and Rodríguez, J.M., 2009. Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR. Letters in Applied Microbiology 48: 523-528. Denman, S.E. and McSweeney, C.S., 2006. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiology and Ecology 58: 572-582. Furet, J.P., Firmesse, O., Gourmelon, M., Bridonneau, C., Tap, J., Mondot, S., Doré, J. and Corthier, G., 2009. Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiology and Ecology 68: 351-362. Haarman, M. and Knol, J., 2006. Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Applied and Environmental Microbiology 72: 2359-2365. Štšepetova, J., Sepp, E., Kolk, H., Lõivukene, K., Songisepp, E. and Mikelsaar, M., 2011. Diversity and metabolic impact of intestinal Lactobacillus species in healthy adults and the elderly. British Journal of Nutrition 105: 1235-1244. Zwielehner, J., Liszt, K., Handschur, M., Lassl, C., Lapin, A. and Haslberger, A.G., 2009. Combined PCR-DGGE fingerprinting and quantitative-pcr indicates shifts in fecal population sizes and diversity of Bacteroides, bifidobacteria and Clostridium cluster IV in institutionalized elderly. Experimental Gerontology 44: 440-446. 6