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.

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Mabel Marian Fisher
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1 Supplementary online material of Beneficial Microbes DOI: 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) , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,698 1

2 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 Saccharomyces DSM Bifidobacterium ATCC bayanus breve Bacteroides fragilis ATCC Enterococcus canis DSM Staphylococcus ATCC aureus Streptococcus salivarius subsp. thermophilus DSM Escherichia coli ATCC Lactococcus lactis subsp. hordniae DSM L. helveticus CGMCC L. helveticus CGMCC 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 L. gasseri ATCC

3 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 L.buchneri DSM L. sakei subsp. sakei DSM L. acidophilus DSM (Haarman & Knol, L. casei DSM (Haarman & Knol, L. delbrueckii subsp.indicus DSM L. paracasei subsp. paracasei DSM 5622 L. plantarum subsp. Argentoratensis DSM (Haarman & Knol, (Haarman & Knol, (Haarman & Knol, L. reuteri DSM (Haarman & Knol, 3

4 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± , Fungi 2.93± , Bacteroides 4.13± , E. coli 4.12± , Enterococcus 3.85± , Staphylococcus 5.03± , Streptococcus 5.77± , Bifidobacterium 3.46± , Lactobacillus 6.34± , Lactococcus 4.12± , lactis 6.16 L. salivarius 2.31± , L. sake 2.43± , L. ruminus 2.73± , L. reuteri 3.95± , L. plantarum 2.76± , L. paracasei 3.38± , L. helveticus 3.90± , High SCC group Mean Median, 8.77± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , Faeces Response Low SCC group P-value of high Mean Median, SCC group ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , High SCC group Mean Median, 11.84± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , Response P-value of high SCC group

5 L. gasseri 3.65± , L. delbrueckii 2.90± , L. casei 3.76± , L. buchneri 3.41± , L. acidophilus 4.08± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ± , ,, 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

6 Table S4. Spearman correlation between individual faecal Lactobacillus species and somatic cell count. Lactobacillus r P-value species L. buchneri L. salivarius L. casei L. sake L. gasseri L. ruminis L. reuteri L. helveticus L. acidophilus L. paracasei L. delbrueckii L. plantarum References Collado, M.C., Delgado, S., Maldonado, A. and Rodríguez, J.M., Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR. Letters in Applied Microbiology 48: Denman, S.E. and McSweeney, C.S., Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiology and Ecology 58: Furet, J.P., Firmesse, O., Gourmelon, M., Bridonneau, C., Tap, J., Mondot, S., Doré, J. and Corthier, G., Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiology and Ecology 68: Haarman, M. and Knol, J., Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Applied and Environmental Microbiology 72: Štšepetova, J., Sepp, E., Kolk, H., Lõivukene, K., Songisepp, E. and Mikelsaar, M., Diversity and metabolic impact of intestinal Lactobacillus species in healthy adults and the elderly. British Journal of Nutrition 105: Zwielehner, J., Liszt, K., Handschur, M., Lassl, C., Lapin, A. and Haslberger, A.G., 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:

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