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Infections with Staphylococcus Aureus and Streptococcus Agalactiae

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Infections with Staphylococcus Aureus and Streptococcus Agalactiae 15/11/2017 1 Association between teat skin colonization and intramammary infections with Staphylococcus aureus and Streptococcus agalactiae Line Svennesen (PhD student) Yasser Mahmmod1, Karl Pedersen2, Volker Krömker3 and Ilka C. Klaas1,4 1Section for Animal Production, Nutrition and Health, Department of Veterinary and Animal Sciences, University of Copenhagen 2National Veterinary Institute, Technical University of Denmark 3University of Applied Sciences and Arts, Hannover, Germany 4DeLaval International AB, Tumba, Sweden (present) Line Svennesen 15/11/2017 2 Project - STOPMAST • University of Copenhagen, Aarhus University, DTU and SEGES – funded by Danish Milk Levy Fund • Overall aim: To investigate how contagious mastitis can be controlled in dairy herds Focus of PhD project: • Shedding patterns • Diagnostics • Reservoirs (potential ways of transmission from one cow to another) • Staphylococcus aureus and Streptococcus agalactiae 15/11/2017 3 Background • Staph. aureus and Strep. agalactiae are causing intramammary infections in dairy cattle - leading to impaired milk quality and economic losses for farmer • Contagious, but environmental and extramammary reservoirs have been described in recent years • Relationship between teat skin colonization and IMI for environmental bacteria • Role of teat skin colonization with Staph. aureus is discussed • Role of teat skin colonization is unknown for Strep. agalactiae • Never investigated in AMS herds with up to 60 cows milked with same unit 15/11/2017 4 Aim • To investigate the association between teat skin colonization and IMI with Staph. aureus or Strep. agalactiae in AMS herds Hypothesis • Colonization of the teat skin increases the risk of a quarter being infected with the same pathogen (Staph. aureus or Strep. agalactiae) 15/11/2017 5 Materials and Methods - Selection • Positive Strep. agalactiae status on bulk tank milk 2016. • ≥ 3 AMS/VMS units • Willing to participate (n=8) • Before sampling (2017): 3 bulk tank milk samples PCR tested (Mastit4, DNA Diagnostic) • Positive in 2 of 3 samples • Ct < 32 for Strep. agalactiae • We expected Staph. aureus in all herds • 30-40 cows >200,000 cells/mL at last milk recording (higest prevalence?) • Milk recording 5-33 days before visit • Randomly selected 15/11/2017 6 Sampling • All quarters per cow • Teats cleaned with dry paper towel • Teat skin sample • Wet-dry method (Paduch & Krömker, 2011) with rayon swabs and 2 mL ¼ Ringer’s solution, 360 degrees rotation • Milk sample • Aseptically collected • NMC standards 15/11/2017 7 Bacterial culture • Samples were homogenized and cultured on: • Edwards medium • Calf blood agar • SA Select medium • Teat skin samples: 100μl (pipette) inoculated and spread onto a whole plate • Milk samples: 10 μl (loop) streaked onto a quarter of a plate Incubated 48 hours at 37 degrees (aerobically). Reading after 24h and 48h 15/11/2017 8 Bacteriological culture: Confirmation • Positive sample: Milk ≥ 1 CFU/10µL, Teat ≥ 1 CFU/100µL (2 mL Ringer’s solution) • Suspected colonies were subcultured • Verification of identification with MALDI-TOF or agglutination test kit Teat skin sample on SA Teat skin sample on Calf Blood agar with 1 Milk sample on Edwards medium Select medium with Staph. colony of Staph. aureus (center, double Positive and negative with Strep. agalactiae (all quarters, aureus (pink colonies) haemolysis) agglutination test clear haemolysis) 15/11/2017 9 Results • 300 cows, 1142 quarters Background of included herds Median values of included cows Herd ID Herd Number of Milk SCC2 No of No of cows Herd ID Parity Days in SCC/mL size1 milking production (cells/mL) cows sampled / (% milk units (ECM/ with of cows with cow/year) high SCC high SCC) H1 2 210 305,000 H1 267 4 10,973 183,000 43 29 (67 %) H2 2 168 554,000 H2 198 3 11,098 211,000 43 37 (86 %) H3 2 182 512,000 H3 344 7 10,733 216,000 74 39 (52 %) H4 2 137 564,000 H4 298 5 11,412 199,000 60 38 (63 %) H5 2 141 710,000 H5 218 4 9,024 255,000 49 39 (80 %) H6 3 158 381,000 H6 247 4 11,701 252,000 59 40 (68 %) H7 3 200 622,000 H7 333 6 11,909 192,000 50 38 (76 %) H8 3 243 515,000 H8 244 4 11,020 338,000 79 40 (51 %) Overall 2 182 502,000 1Includes both lactating and dry cows. 2Geometric mean of bulk tank SCC within last three months. Median 15/11/2017 10 Results – Number of positive quarters by herd Staph. aureus no of quarters positive by herd Strep. agalactiae no of quarters positive by herd Staphylococcus aureus Streptococcus agalactiae Herd Ct-value Herd Ct-value Teat Milk n Teat Milk n bulk milk bulk milk H1 29 - 32 1 18 111 H1 23 - 40 0 1 111 H2 25 - 40 1 4 145 H2 22 - 32 0 0 145 H3 33 - 40 14 3 148 H3 25 - 29 0 10 148 H4 21 - 40 0 3 144 H4 21 - 25 3 10 144 H5 28 - 32 14 3 149 H5 20 - 26 0 17 149 H6 26 - 40 10 2 154 H6 25 - 40 0 3 154 H7 24 - 29 25 8 141 H7 24 - 25 0 29 141 H8 27 - 31 10 52 150 H8 21 - 24 1 14 150 Total Total 75 93 1142 4 85 1142 Interesting differences between herds – related to management? Strain types? 15/11/2017 11 Results – Staphylococcus aureus • Overall prevalences (high SCC cows) • Staph. aureus milk: 8.1 % • Staph. aureus teat: 6.6 % • Lower than expected (cow level IMI ~20 %) • 3.2 higher odds of a quarter being milk positive and teat skin positive, relative to teat skin negative and milk positive 15/11/2017 12 Results – Streptococcus agalactiae • Overall prevalences (high SCC cows) • Strep. agalactiae milk: 7.4 % • Strep. agalactiae teat: 0.35 % • Cow level IMI ~20 % • First time Strep. agalactiae was isolated from teat skin • In 4 quarters (2 cows) also having Strep. agalactiae in milk. • No association was calculated 15/11/2017 13 Conclusion • Strep. agalactiae can be isolated from teat skin • Only a few Strep. agalactiae cultured from teat skin • Low Se of the method? • Strep. agalactiae does not colonize the teat skin • Herd variation in number of positive samples for both pathogens • Teat skin colonization with Staph. aureus increases odds of IMI with Staph. aureus • No causality in this study (cross sectional) 15/11/2017 14 Further analysis • A subset of samples (all right rear quarters) DNA Diagnostics tested with PCR • More teat skin samples positive for both Staph. aureus and Strep. agalactiae compared to bacterial culture • Will look further into the interpretation of that Thanks to: Nanna Krogh Skjølstrup and Louise Råkjær Mathiasen (Master students) 15/11/2017 15 Thank you! Thanks to: Nanna Skjølstrup and Louise Raakjær (Master students) .
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