Association between teat skin colonization and intramammary infections with Staphylococcus aureus and Streptococcus agalactiae

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1 15/11/ Association between teat skin colonization and intramammary infections with Staphylococcus aureus and Streptococcus agalactiae Line Svennesen (PhD student) Yasser Mahmmod 1, Karl Pedersen 2, Volker Krömker 3 and Ilka C. Klaas 1,4 1 Section for Animal Production, Nutrition and Health, Department of Veterinary and Animal Sciences, University of Copenhagen 2 National Veterinary Institute, Technical University of Denmark 3 University of Applied Sciences and Arts, Hannover, Germany 4 DeLaval International AB, Tumba, Sweden (present)

2 Line Svennesen 15/11/ 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

3 15/11/ 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

4 15/11/ 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)

5 15/11/ Materials and Methods - Selection Positive Strep. agalactiae status on bulk tank milk 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 cows >200,000 cells/ml at last milk recording (higest prevalence?) Milk recording 5-33 days before visit Randomly selected

6 15/11/ 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

7 15/11/ 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

8 15/11/ 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 Select medium with Staph. aureus (pink colonies) Teat skin sample on Calf Blood agar with 1 colony of Staph. aureus (center, double haemolysis) Positive and negative agglutination test Milk sample on Edwards medium with Strep. agalactiae (all quarters, clear haemolysis)

9 15/11/ Results 300 cows, 1142 quarters Background of included herds Herd ID Herd size 1 Number of milking units Milk production (ECM/ cow/year) SCC 2 (cells/ml) No of cows with high SCC No of cows sampled / (% of cows with high SCC) H , , (67 %) H , , (86 %) H , , (52 %) H , , (63 %) H , , (80 %) H , , (68 %) H , , (76 %) H , , (51 %) 1 Includes both lactating and dry cows. 2 Geometric mean of bulk tank SCC within last three months. Median values of included cows Herd ID Parity Days in milk SCC/mL H ,000 H ,000 H ,000 H ,000 H ,000 H ,000 H ,000 H ,000 Overall Median ,000

10 15/11/ Results Number of positive quarters by herd Staph. aureus no of quarters positive by herd Herd Ct-value bulk milk Staphylococcus aureus Teat Milk n H H H H H H H H Total Strep. agalactiae no of quarters positive by herd Herd Ct-value bulk milk Streptococcus agalactiae Teat Milk n H H H H H H H H Total Interesting differences between herds related to management? Strain types?

11 15/11/ 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

12 15/11/ 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

13 15/11/ 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)

14 15/11/ 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 15/11/ Thank you! Thanks to: Nanna Skjølstrup and Louise Raakjær (Master students)