6th Dutch Mastitis Research Workers Meeting

Transcription

1 16 April 2013, Utrecht, The Netherlands

2 Abstracts 16 April 2013 Utrecht, the Netherlands 2013 All rights reserved. No part of this document may be reproduced or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission of the authors. The individual contribution in this publication and any liabilities arising from them remain in the responsibility of the authors Cover design: Felix van Soest Image cover :

3 Table of contents Using volatiles metabolites to identify mastitis pathogens from science to practice Kasper Hettinga, Frank de Bok and Theo Lam Consequences of farmers interpretation of mastitis alerts K.J. Buma, R. Jorritsma and H. Hogeveen Molecular typing of S. aureus from goat milk samples: preliminary results G. Koop, T.J.G.M. Lam, V.O. Baede and J.A. Wagenaar Management practices associated with achieving milk quality premiums in Flemish dairy herds. Pieter Passchyn, Sofie Piepers and Sarne De Vliegher Host response during experimental intramammary infection with S. chromogenes and S. fleurettii: preliminary results Kristine Piccart, Joren Verbeke, Sofie Piepers and Sarne De Vliegher Factors associated with the probability of diagnosing intramammary infections in dairy cows Sofie Piepers, Reshat Jashari and Sarne De Vliegher Evaluation of dry cow therapy in low somatic cell count cows Christian Scherpenzeel The relative importance of management measures to improve udder health in relation to other farm management measures when extra time would become available description of methodology. Felix van Soest, Monique Mourits and Henk Hogeveen Extended treatment versus standard treatment of clinical mastitis. Jantijn Swinkels Sustainable dairy farming by responsible use of veterinary medicines: preliminary results Marina Stevens, Sofie Piepers, Sarne De Vliegher Evaluation of the Rinsing of Teat Cup Liners with Cold Water, Hot Water or Steam. Dimitri Valckenier, Sofie Piepers, Sarne De Vliegher Cow and quarter level risk factors for IMI with the more pathogenic CNS species: outline of a new study and preliminary results Anneleen De Visscher, Freddy Haesebrouck, Sofie Piepers, Sarne De Vliegher Clinical mastitis incidence in Flanders Joren Verbeke, Sarne De Vliegher

4 Using volatiles metabolites to identify mastitis pathogens from science to practice Kasper Hettinga 1, Frank de Bok 2, Theo Lam 3 1 Wageningen University, Wageningen 2 KBBL Wijhe BV 3 Animal Health Service, Deventer In Hettinga et al. (2008), we proposed a new method for mastitis pathogen identification. This method is based on the detection of volatile metabolites produced by mastitis pathogens. The metabolites are analysed using gas chromatography/mass spectrometry (GC/MS). The pattern of volatile metabolites is then analysed using an artificial neural network, resulting in pathogen identification. This method, by relying on GC/MS is, however, not suitable for large scale application, due to the labour intensity and costs associated with this method. The aim of this study was therefore to test whether a simpler approach could still lead to successful pathogen identification. Materials & methods Samples were either prepared by inoculating mastitis pathogens in raw milk from healthy cows, or were obtained from the diagnostic sample stream. Samples were pre-incubated in milk before analysis. Details of the existing method can be found in Hettinga et al. (2008). Several simplifications were tested compared to the original setup: 1) testing different GC columns to circumvent the use of liquid nitrogen (increases speed and reduces costs), 2) testing reduced pre-incubation times before analysis (In a previous study on the influence of incubation time (Hettinga et al, 2009), it was concluded that at least 8 hours of incubation would be needed, but an incubation period between 4 and 8 hours was not assessed), 3) using a regular GC instead of GC/MS (simpler & cheaper). Results & discussion Different GC column Using a more polar GC column compared to the previous published method led to a simplification of the analytical approach, with an improved quality of the data. This adaptation was therefore used for all analyses within the project. Reduction of incubation time For most samples, 0 and 4 hours of incubation was too short for noticeable volatile metabolite formation, which corresponds to the findings report in Hettinga et al (2009). For samples obtained from the diagnostic sample stream, predicting the pathogen present was equally successful in samples incubated 6 and 8 hours. Regular GC instead of GC/MS Using a regular GC, without using the mass spectrometer for identification, led to problems with components that could not be separated anymore due to overlapping retention times in the GC. The same data analysed using regular GC/MS led to results similar to Hettinga et al (2008). Conclusions The results from this study indicate that the use of a more polar column is advantageous for analyzing volatile metabolites, as it is easier and it yields higher quality data. The incubation time required for volatile metabolite formation may be reduced to 6 hours, allowing the whole analysis to be finished within one working day. However, the use of regular GC instead of GC/MS is not possible. The complexity and cost of a GC/MS system are such that this limits the application of the method in practice. References Hettinga KA, van Valenberg HJF, Lam TJGM, van Hooijdonk ACM Detection of mastitis pathogens by analysis of volatile bacterial metabolites. J Dairy Sci. 91(10): Hettinga KA, van Valenberg HJF, Lam TJGM, van Hooijdonk ACM The influence of incubation on the formation of volatile bacterial metabolites in mastitis milk. J Dairy Sci. 92(10):

5 Consequences of farmers interpretation of mastitis alerts K.J. Buma, R. Jorritsma and H. Hogeveen Department of Farm Animal Health Faculty of Veterinary Medicine When milking cows with an automatic milking system (AMS), for mastitis detection, farmers have to rely on sensor alerts. It is known that, although the sensitivity and specificity of current mastitis are quite reasonable, the systems still give a relative large number of false positive alerts. In order to find detect all mastitis cases, farmers have to visually check all alerts. We do know that far from all farmers are doing that. The goal of this research was therefore to study farmers behavior related to mastitis alerts and the consequences thereof. The study was done on 7 farms, located around one village in the north of the Netherlands. The farmers needed to be motivated to enter the study. All farms were milking with a Lely Astronaut (Lely, Rotterdam, the Netherlands) AMS. Each farm was visited five times. During each visit, the farmers were questioned about the alerts on the alert list of that day. After this questioning, the milk of all four quarters of every cow on the alert list was visually checked for clinical mastitis and was checked for subclinical mastitis using the CMT test. During the first visit and before checking individual quarters, a questionnaire was held on the criteria farmers used for selecting cows on the alert list to be visually checked. Important criteria for farmers were, an alarming change in milk production, flakes and/or clots on the milk filter in combination with high electrical conductivity or a failed milking. Reasons for not checking alerts were: no flakes or clots on the milk filter, no change in milk production, cows that were repeatedly on the alert list or a lack of time. In total 421 alerts were studied (Table 1). Of all mastitis alerts, 60 % were not associated with mastitis. For the repeated alerts (cows that had been on the alert list earlier), 46 % of the alerts were not associated with mastitis. Only 15 of the 421 quarter alerts were checked by the farmer (3 % of all alerts). From the checked alerts by the farmers, 67 % had clinical mastitis and 13 % had subclinical mastitis. However, the farmers missed quite a large number of mastitis cases. Of the 39 clinical mastitis cases that were found, only 10 were detected by the farmers, which means that 74 % of the clinical mastitis cases were not (yet) detected by the farmers. In conclusion, in order to detect all clinical mastitis, farmers should check all alerts visually. For the 7 farmers in this study this would mean that, on average, 12 cows need to be checked each day. Table 1 Studies alerts and mastitis 5

6 Genotyping of Staphylococcus aureus in dairy goats G. Koop, 1,* T.J.G.M. Lam, 1,2, V.O. Baede, 3 J.A. Wagenaar 3 1 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, P.O. Box , 3508 TD Utrecht, The Netherlands 2 GD Animal Health Service, P.O. Box 9, 7400 AA Deventer, The Netherlands 3 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box , 3508 TD Utrecht, The Netherlands Staphylococcus aureus is an important mammary pathogen in goats and responsible for the majority of severe clinical mastitis cases (Bergonier et al., 2003; Smith and Sherman, 2009). To prevent clinical mastitis, understanding the transmission of this pathogen is important. Molecular typing of pathogens can be used to determine whether contagious or environmental transmission is a more likely route. Little variation within strains cultured from one herd is an indication of clonal spread and thus of either a point source infection, or of contagious transmission, whereas environmental transmission is generally characterized by a great diversity of strains cultured within a herd. The goal of this study was to assess the amount of variation in S. aureus isolates within and between herds and within and between countries. Materials, methods, results and discussion We performed a systematic literature search for S. aureus isolates from sheep and or goats that had been genotyped with either surface protein A (spa)-typing or multi-locus sequence typing (MLST). This resulted in an overview of the most common spa- and sequence types (ST) of S. aureus in small ruminants. The MLST sequence type ST133 was the most common among goat isolates (39%), but a number of other ST s had also been reported. Furthermore, we collected S. aureus from clinical and subclinical cases of mastitis in goats in The Netherlands. Spa-typing was performed on these isolates and a selection of isolates was also characterized by Pulsed Field Gel Electrophoresis (PFGE). This showed a large similarity between Dutch goat S. aureus, within farms as well as between farms. Most of the isolates belonged to the same sequence type ST133. Some isolates belonged to ST398. To study genetic variation in goat S. aureus from other continents, we collected isolates from California and New Zealand. These isolates are currently being spa-typed and results are not available yet but will probably be presented at the DMRW annual meeting. So far, this study shows that worldwide a single S. aureus sequence type seems to be dominant within goats, which is also highly prevalent within the Netherlands. In Dutch isolates, a high degree of similarity between isolates was observed, and contagious spread seems to be the most probable route of transmission. Furthermore, a high homology was found between S. aureus from clinical mastitis and from subclinical mastitis, indicating that reducing the prevalence of subclinical S. aureus infection may help reduce the incidence of clinical mastitis. References Bergonier, D., de Cremoux, R., Rupp, R., Lagriffoul, G., Berthelot, X., Mastitis of dairy small ruminants. Vet. Res. 34, Smith, M.C., Sherman, D.M., Chapter 14: Mammary gland and milk production. Goat medicine. Wiley-Blackwell, Ames, Iowa, pp

7 Management practices associated with achieving milk quality premiums in Flemish dairy herds. Pieter Passchyn 1,2, Sofie Piepers 2, Sarne De Vliegher 2 1 Milk@vice, Torhout, Belgium 2 M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium In Flanders, achieving good milk quality is encouraged by a system including both milk quality premiums and milk quality penalties for the farmers. Both are based on the bulk milk somatic cell count (BMSCC), the total bacterial count (TBC) and the coliform count (CC), besides other legal standards as the freezing point, the filtration test and the absence of residues in any milk delivery. Management practices associated with BMSCC have been evaluated in several studies. Still, the number of studies describing risk factors for TBC and CC is limited. The aim of our study was to determine to what extent differences in management practices were associated with achieving the milk quality premium in one year. As a second objective, we wanted to measure the economic value of reaching the milk quality standards in Flanders. Materials and Methods A web-based survey was conducted on 242 dairy herds in Flanders. The survey contained 41 main questions all concerning the year 2009 and covered several topics: general management, herd health management, milking routine, calving and dry cow management, and nutrition. Monthly measured SCC, TBC and CC values were available for the year 2009 for all herds. Statistical analysis was done using binary logistic regression with achieving a milk quality premium during all 12 months (yes/no) as outcome variable. Results Fifty-six percent (n = 135) of the farmers did achieve their milk quality premium the whole year around. In almost 87% of the months (n = 2904 months), all herds achieved the milk quality standards. The most common reason for not achieving the premium was an elevated CC. The average lost revenue per cow per year was 17.7 (SD = 16.2) based on herd size and the number of months the premium was not achieved. Univariable analysis (p < 0.15) resulted in 17 management practices being associated with the outcome variable. Multivariable analysis through stepwise reduction of the model, including 2-way interactions, resulted in 4 management practices that were significantly associated with achieving a milk quality premium during all 12 months (P< 0.05). Feeding dry minerals (OR = 1.95; ) and predipping/prespraying (OR = 2.98; ) increased the likelihood of achieving the milk quality premium throughout The likelihood of achieving the premium was reduced when cows were milked with an automated milking system (AMS) (OR = 0.044; ) and when the monthly clinical mastitis incidence was higher than 3% (OR = 0.47; ). Conclusions Almost 50% of the Flemish dairy herds included in the study did not achieve their milk quality premium every month, with an elevated CC being the main reason. Achieving the milk quality standards is driven by day-to-day management (e.g. clinical mastitis, hygiene, immunity) but also by long-term decisions (e.g. installing an AMS). Farmers working with AMS need to be aware of the fact that reaching the required milk quality standards to obtain the milk quality premium might be more difficult. 7

8 Host response during experimental intramammary infection in dairy heifers with S. chromogenes and S. fleurettii - preliminary results Kristine Piccart, Joren Verbeke, Sofie Piepers, Sarne De Vliegher M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria in subclinical bovine mastitis cases. Conventionally, CNS are considered as one large bacterial group even though roughly a dozen different species are found in milk. Although CNS are generally regarded as minor pathogens, little information is available on the host-pathogen interaction of individual species. Previous research even demonstrates a positive association between CNS infection and milk production in heifers and multiparous cows. The aim of this study is to elucidate the immune response of dairy heifers after experimental infection with Staphylococcus chromogenes, a so-called udder-adapted species, and Staphylococcus fleurettii, a so-called environmental species. Materials and Methods Eight clinically healthy Holstein-Friesian heifers in mid-lactation are randomly selected for an experimental infection with 3 different bacterial isolates: (1) S. chromogenes: a strain isolated from a chronically infected cow (2) S. chromogenes: a strain that inhibits the growth of major pathogens in vitro (3) S. fleurettii: a strain isolated from sawdust bedding A split-udder design is used in which three quarters of the uninfected heifers are inoculated with 1 x 106 colony-forming units (cfu) of the aforementioned bacteria and one quarter is infused with 5 ml sterile phosphate buffered saline, thereby serving as a control. During the study, the heifers are kept in a tie-stall barn and monitored for five days. The somatic cell count (SCC) and bacterial elimination rates are quantified repeatedly. Furthermore, the proportion of viable neutrophils in milk is determined using a double staining technique with annexine V-fluorescein isothiocyanaat (FITC) and propidium iodide. Milk production is also measured at quarter level. Milk and blood samples are collected for further analysis of prolactin levels, several pro-inflammatory cytokines (TNF-α, IL-1β, IL-8, GM-CSF) and serum amyloid A. The CNS isolates found in milk are stored at -80 C for later confirmation by means of tdna-pcr and strain typing. Preliminary results Based on the descriptive results of 4 heifers, it appears that the chronic S. chromogenes strain remains in the udder for approximately 48 hours, causing the highest and longest increase of the quarter milk SCC. The other S. chromogenes strain and S. fleurettii are eliminated within 28 hours. None of the heifers showed any signs of clinical mastitis. 8

9 Factors associated with the probability of diagnosing intramammary infections in dairy cows Sofie Piepers, Reshat Jashari, Sarne De Vliegher M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium To encourage farmers and veterinarians to sample cows for bacteriological culturing, the probability of isolating a mastitis pathogen from the collected milk samples should be maximized. Obviously, this probability is related to the cows test-day cscc and the specific SCC-threshold that is been used. The objective of this study was to determine to what extent the test-day cscc and other factors such as the herd prevalence of intramammary infections, parity and days in milk (DIM) affect the probability of diagnosing IMI with different pathogens in dairy cows. Materials and Methods A total of 780 single quarter milk samples from 195 dairy cows were collected for bacteriological culturing at 21 cross-sectional herd screenings performed as part of different research projects. Based on their quarter milk IMI-statuses, cows were classified as non-infected, infected with any pathogen or infected with a major pathogen. Additionally, monthly composite milk samples from all lactating cows were available during four successive milkings with an interval between 4 and 6 weeks preceding the herd screening (Flemish Cattle Breeding Association, Oosterzele, Belgium). Logistic mixed regression models were fit to determine the association between parity, average test-day herd SCC (hscc) as an indicator for the herd prevalence of IMI, DIM at sampling, and cscc at the test-day closest to the herd screening and the probability for diagnosing IMI (MLwiN 2.02, Center for Multilevel Modeling, Bristol, UK). The binary outcome variables were (1) IMI with any pathogen (0 = non-infected; 1 = infected with any pathogen), and (2) IMI with a major pathogen (0 = non-infected; 1 = infected with a major pathogen). The predictor variables hscc and cscc were categorized as < 200,000 cells/m vs 200,000 cells/ml. In all models, herd was included as random effect to correct for clustering of cows within herds. Using a prediction equation, the probability for diagnosing IMI with either any pathogen or a major pathogen was calculated as followed: P = 1/[1 + exp(-1 x (intercept + βparity + βdim+ βcscc + βhscc)]. Results As was expected, the probability of diagnosing IMI with any pathogen was highest when the average test-day hscc was > 200,000 cells/ml, and the cscc at the test-day closest to bacteriological culturing was > 200,000 cells/ml. The probability was lowest for animals with a test-day cscc 200,000 cells/ml and kept on herds with an average test-day hscc 200,000 cells/ml [49.5% - 95% confidence interval (95%CI) 42.5% 56.5%] and highest for animals with a cscc > 200,000 cells/ml kept on herds with an average test-day hscc > 200,000 cells/ml (94.4% - 95%CI 91.1% 97.6%). For IMI with a major pathogen, the probability also increased by parity (> 1 lactation) and varied between 2.6% (95%CI 0.0% - 5.7%) for heifers with a cscc 200,000 cells/ml and kept on herds with a test-day hscc 200,000 cells/ml and 51.8% (95%CI 41.9% 61.7%) for multiparous cows with a cscc > 200,000 cells/ml and kept on herds with a test-day hscc > 200,000 cells/ml. Conclusions The probability of isolating a mastitis pathogen from dairy cows depends on the mastitis pathogen that one is focusing on with the probability of diagnosing IMI with any pathogen being higher than the probability of diagnosing IMI with a major pathogen. To further fine-tune the selection of cows for bacteriological culturing, it seems to be recommended to not only take into account the test-day cscc, but also the average test-day hscc and parity in particular for the identification of cows most likely infected with a major pathogen. 9

10 Evaluation of dry cow therapy in low somatic cell count cows C.G.M. Scherpenzeel 1, I. den Uijl 1, G. van Schaik 1, J. Keurentjes 1, T.J.G.M. Lam 1,2 1 GD Animal Health Service, PO Box 9, 7400 AA Deventer, The Netherlands 2 Utrecht University, Department Farm Animal Health, PO Box 80151, 3508 TD Utrecht, The Netherlands. The goal of dry cow therapy is to reduce the level of intramammary infection by eliminating existing infections at drying off and preventing new infections to occur during the dry period (Bradley and Green, 2001). Preventive use of antibiotics, however, is questionable due to public health concerns, because selective pressure on bacterial populations can contribute to development of antimicrobial resistance (Tacconelli, 2009) (Landers et al., 2012). This study aimed to evaluate dry cow therapy in 1657 cows on 97 Dutch dairy herds, that all had a low somatic cell count (SCC) at the last milk production recording before drying off. A split-udder design was used in which two quarters of each cow were treated with dry cow antibiotics and the other two quarters remained as untreated controls. The effects of dry cow therapy on clinical mastitis, bacteriological status, somatic cell count and antibiotic use were determined on quarter level using logistic regression and chi-square tests. Results show that the incidence rate of clinical mastitis in untreated quarters was higher than in treated quarters, with Streptococcus uberis as the predominant organism causing clinical mastitis in both groups. Somatic cell count of the untreated quarters was significantly higher than the treated quarters at calving and at 14 days in lactation. The use of antibiotics on herd level was reduced. We concluded that selective dry cow therapy, not giving dry cow antibiotics to low SCC cows, leads to more cases of clinical mastitis and higher cell counts in these cows. At the same time, antibiotic use decreased substantially. References Bradley, A. J. and M. J. Green An investigation of the impact of intramammary antibiotic dry cow therapy on clinical coliform mastitis. Journal of dairy science 84(7): Landers, T. F., B. Cohen, T. E. Wittum, and E. L. Larson A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep 127(1):4-22. Tacconelli, E Antimicrobial use: risk driver of multidrug resistant microorganisms in healthcare settings. Current opinion in infectious diseases 22(4):

11 The relative importance of management measures to improve udder health in relation to other farm management measures when extra time would become available Description of methodology Felix van Soest 1, Monique Mourits 1 and Henk Hogeveen 1,2 1 Chair Group Business Economics, Wageningen University 2 Department of Farm Animal Health, Utrecht University A total of 56 French, 60 German, 28 Spanish and 56 Swedish organic dairy farmers were approached to complete an adaptive conjoint analysis (ACA) concerning their preference toward different farm management areas. The farm visits followed a strict protocol and were carried out by one or two persons per country. The farm visits lasted approximately 2 hours of which the adaptive conjoint analysis lasted approximately ½ hour, the welfare quality assessment 1 hour and the general questionnaire on farm characteristics ½ hour and took place from March 2013 until June In this study the conjoint model is used to value the preference of farmers for different farm management areas when extra time would become available. Farm management when extra time becomes available is here the product and different farm management areas are the attributes of this product. Methodology of the current study was based on the study of Huijps et al. (2009) were perception of farmers towards udder health was determined using ACA. Five different management areas where chosen in such a way they were independent of each other. The following management areas were defined: udder health management, barn management, claw health management, calf management and pasture management. As an indicator for every farm management area, three related management measures were described (Table 1). Management area and management measures were based on literature reviews, experts opinions and farmers input. Due to the design of the questions ( when extra time becomes available ) the constructed management measures had to fulfil three criteria: 1) not likely to be currently performed on dairy farms, 2) had to be suggested improvements for the management area and 3) could be performed by the farmer himself. Design of the management measure had to be as strict as possible leaving little room for own interpretation. The farmers were asked to judge their preference for measures when extra labour time would become available. While choosing levels as strict as possible the ACA leaves room for own evaluation on possible efficiency, costs of the management measures and the area of interest to improve thereby representing more real life situation when implementing management measures when extra time becomes available. Table 1 overview of farm management areas and management measures References Huijps, K., Hogeveen, H., Lam, T.J.G.M., Huirne, R.B.M., Preferences of cost factors for mastitis management among Dutch dairy farmers using adaptive conjoint analysis. Preventive Veterinary Medicine 92,

12 Extended treatment versus standard treatment of clinical mastitis Jantijn Swinkels GD Animal Health Service, P.O. Box 9, 7400 AA Deventer, The Netherlands Mastitis is a costly disease on dairy farms and should therefore be controlled. Efficient mastitis treatment protocols are part of mastitis control. Extended treatment of clinical mastitis has been advocated as it is perceived to be more efficient than standard treatment. In the EU, such extended treatment protocols are usually off-label and can only be justified if evidence based. To collect evidence, 3 large clinical trials were set up throughout Europe. All 3 trials compared extended 5-day intramammary cefquinome treatment to 1.5-day intramammary cefquinome treatment of clinical mastitis. One trial was conducted in 5 EU countries (F, NL, Hu, UK, It) and compared the 2 treatment protocols on clinical S. aureus mastitis (n=206). Another was performed in the UK on 3 farms with high recurrence of environmental, E. coli and S. uberis, mastitis(n=994). The last trial was performed in Germany looking for the efficacy on farms with predominantly S. uberis mastitis (n=409). Extended treatment protocols of clinical mastitis did not improve overall bacteriological cure compared to standard treatment in any of the 3 trials. However, there was a strong trend for extended treatment to be beneficial for clinical S. uberis mastitis. Extended treatment improved clinical cure of S. aureus, resulted in less clinical persistence or recurrence in the UK, whilst the German trial showed the disappearance of clinical signs simply needs time. In conclusion, extended treatment of clinical mastitis did not improve bacteriological cure on average and should be discouraged for routine use. We found indications that after standard treatment, the disappearance of clinical signs simply needs time and not antibiotics. At the pathogen level, we showed a strong trend towards extended treatment of S.uberis clinical mastitis being beneficial suggesting such protocols should be restricted to farms where S. uberis predominates in the aetiology of clinical mastitis. 12

13 Quantification of the use of antimicrobials on dairy farms in Flanders, Belgium Marina Stevens, Sofie Piepers, Sarne De Vliegher M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium Antimicrobials are indispensable tools for decreasing the prevalence and incidence of infectious diseases and their use in veterinary medicine has a positive effect on animal health, well-being and productivity. Still, the society demands accountability when using and/or selling veterinary antimicrobials especially in food producing animals. Excessive and/or injudicious use of antimicrobials is seen as a health threat as it potentially promotes the development of antimicrobial resistance of veterinary and human-associated bacteria. The dairy industry has a well-respected image when it comes down to safeguarding the quality of its products, based on substantial testing of multiple milk quality parameters, residue testing,. Still, data quantifying and qualifying the use of antimicrobials are lacking. Materials and Methods A project was designed to (1) quantify the antimicrobial use (AMU) on Flemish dairy farms, (2) to determine the association between AMU and the prevalence of antimicrobial resistance of mastitis pathogens cultured from milk samples, (3) to study how the AMU is related to the udder health status of the herd, and (4) to evaluate to what extent the AMU and the prevalence of antimicrobial resistance can be reduced via proactive udder health and milk quality counselling. The latter is possible as half of the included herds (n = 30) are being visited on a regular basis to work on udder health and milk quality, whereas the other half serve as controls (n = 31). Data are collected on sixty-one dairy farms distributed over the 5 different Flemish provinces. The average herd size varies between 40 and 130 lactating dairy cows. The usage of antimicrobials was recorded between January 2012 and September 2012 through so-called garbage can audits on all participating herds. Farmers were instructed to deposit all empty drug containers used for the treatment of adult lactating and dry dairy cows in the receptacles. In September 2012, the empty drug containers were collected and counted and the AMU data were entered in an Excel-file. The antimicrobial drug use rate (ADUR), defined as the number of animal daily doses (ADD) used on a farm per cow per year, was calculated. Bulk milk somatic cell count records measured at a weekly-interval during the study period were retrieved from the Milk Control Centre Flanders and the participating farmers were asked to record every case of clinical mastitis. Also, at the onset of the study, quarter foremilk samples of ten cows per farm suspected of being subclinically infected were collected for bacteriological culturing and antimicrobial sensitivity testing. This will be repeated at the end of the study. Results and Conclusions Preliminary results indicate an average herd ADUR in Flanders of 8.4 ADD/cow/year with a median value of 7.3. A large variation among the different herds was evident from the range between 1.2 up and 20.4 ADD/ cow/year. The highest proportion of AMU was used for intramammary treatments (5.5 ADD/cow/year) of which 53% for antimicrobial dry cow therapy. Beta-lactam antibiotics have the highest ADUR (6.5 ADD/ cow/year) and the antimicrobial drugs of high importance in human medicine, i.e. the 3th and 4th generation cephalosporins and fluoroquinolones, account for more than 25% of the total AMU, which is worrying. In the upcoming months, collection of AMU data will continue, and the gathered AMU data will be explored further. In addition, the association between the herds udder health status and the AMU on the one hand and the association between AMU and the prevalence of antimicrobial resistance of mastitis pathogens on the other hand will be determined. 13

14 Evaluation of the rinsing of teat cup liners with cold water, hot water or steam - preliminary results Dimitri Valckenier, Sofie Piepers, Sarne De Vliegher M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium Mastitis is still a disease with major consequences for the dairy sector and a huge impact on the profitability of dairy farms. Contagious bacteria are primarily transferred from one cow to another during the milking process preferably via the teat cup liners. Cleaning of contaminated teat cup liners during the milking process might be an important preventive measure in limiting the risk of spread of those bacteria. In this trial, the effectiveness of three different rinsing protocols in reducing the number of bacteria in the teat cup liners was evaluated. Materials and Methods This study was conducted on a Flemish dairy farm with approximately 90 lactating Holstein-Friesian cows. The cows were housed in a free stall with cubicles and milked on a 22-stand rotary milking parlour. After milking and removal of the teat cups, a sample was taken from the inside of each teat cup with a cotton wool swab. The first 10 milking clusters were then treated for three seconds with hot water (±70 C), the next 10 with steam using a portable steam generator, and the following 10 with cold water (±10 C). Immediately after treatment, another sample of the teat cup liners was taken. In addition 7 milking clusters were sampled twice and served as a negative control group. The swabs were stored at 4 C before further processing. Each sample was dissolved in 2 ml PBS and diluted (10-1, 10-2 and 10-3). Subsequently, 10 μl of each dilution was streaked on Columbia agar with 5% sheep blood (Oxoid, Basingstoke, UK). After 24 hours of aerobic incubation at 37 C, all colonies were counted and expressed as cfu/ml. A log10-transformation of cfu/ml was applied to normalize the data. As almost no colonies were grown on the 10-3 dilution, only the results obtained at the 10-1 and 10-2 dilution were included in the analyses. All data were analysed with SAS Enterprise Guide 4 using a linear mixed regression model. The variable teat cup liner was included as random effect to account for the clustering of repeated samplings within a teat cup. The model with log10 cfu/ml as outcome variable included treatment (1 = hot water; 2 = steam; 3 = cold water; 4 = no treatment), period (1 = before treatment; 2 = after treatment), and the interaction term between both variables. Results The repeatability of the sampling technique was evaluated by calculating the difference in cfu/ml between the two consecutive samples collected from the teat cup liners in the negative control group. As the average log10cfu/ml only marginally differed between both samplings (2.4% - P = 0.74), differences in cfu/ml between the samples collected before and after treatment in the other groups could not be due to the way of sampling. Rinsing of the teat cup liners with cold water resulted in a significant reduction (P < 0.001) of the number of back-transformed cfu/ml with 39.5%. Hot water and steam resulted in a reduction of 60.4% (P < 0.001) and 72.4%, respectively (P < 0.001). However, from the results of this study, it could not be shown that the reduction by steam is significantly higher than the reduction by hot water (P = 0.25). Conclusions In this pilot study, rinsing of the teat cup liners for three seconds with either cold water, hot water or steam after teat cup removal resulted in a substantial reduction of the number of log10cfu/ml. The largest reduction was obtained by application of the steam or hot water protocol. Importantly, none of these methods resulted in a 100% reduction, suggesting that a total disinfection of teat cup liners during the milking process will be difficult to reach, independently from the method that is applied. However, rinsing of the teat cup liners after teat cup removal can be considered as an effective preventive measure for limiting the spread of bacteria during the milking process. The preferred method to disinfect the teat cup liners during milking does obviously not only depend on the effectiveness but also on practical aspects such as the possibility of installing a water heater or the possibility of purchasing and installing a steam generating devise. 14

15 Cow and quarter level risk factors for IMI with the more pathogenic CNS species: outline of a new study and preliminary results Anneleen De Visscher 1, Freddy Haesebrouck 2, Sofie Piepers 1, Sarne De Vliegher 1 1 M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium 2 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium Coagulase-negative staphylococci (CNS) are the most frequently isolated mastitis pathogens from cows with and without a high somatic cell count (SCC). They are also the major cause of mastitis in heifers. In addition, they are abundantly present in the cow s environment and on teat apices. The bacteria within this heterogeneous group have traditionally been regarded as minor pathogens. We recently reported that Staphylococcus chromogenes, S. xylosus and S. simulans can cause an intramammary infections characterized by elevated quarter somatic cell count to a level that is not different from intramammary infections (IMI) due to Staphylococcus aureus. These CNS species are therefore considered to have more impact on udder health. Research is needed to get more insight in prevention and control measures for IMI with those three species. Therefore, the main objective of this study is to identify cow and quarter level risk factors for intramammary infections at parturition caused by the more pathogenic CNS species. Materials and Methods Twelve cows and heifers on thirteen Flemish dairy farms were enrolled in this study. Fourteen days before parturition, teat apex swabs were taken to determine teat apex colonization. Within four days after parturition and one month later, quarter milk samples were collected. Swabs and milk samples were plated on mannitol salt agar, a suitable medium for the recovery of bovine-associated coagulase-negative staphylococci. All phenotypically different types of colonies were counted, picked-up, plated on aesculin blood agar and subjected to further analysis. All bacteria considered to be CNS were identified to the species level using the molecular technique transfer RNA intergenic spacer PCR. If no identification can be obtained, isolates are subjected to sequencing of the rpob gene. Potential cow and quarter level risk factors for IMI at parturition were recorded at the three sampling times. Preliminary results Teat apex swabs and milk samples from ten randomly selected cows and heifers were evaluated. Teat apices were colonized with S. chromogenes, S. haemolyticus, S. equorum, S. devriesei and S. sciuri. At parturition, IMI were caused by S. chromogenes, S. haemolyticus, S. sciuri and S. vitulinus. One month later, S. chromogenes, S. haemolyticus, S. equorum, S. devriesei and S. cohnii were isolated from some quarter milk samples. S. chromogenes was the only species found on teat apices and in milk samples of the same quarters. Teat apices of five animals were colonized with S. chromogenes. At parturition, S. chromogenes was isolated from milk samples of the corresponding quarters of three of those cows. One month later, only one cow had an intramammary infection with S. chromogenes in the same quarter. Despite the use of an iodide containing teat dip during the dry period, S. chromogenes, S. haemolyticus, S. equorum and S. devriesei were isolated from those teat apices. S. sciuri was only isolated from non-dipped teat apices. Future work All swabs (n = 624) and milk samples (n = 1248) have now been collected and 1550 CNS isolates have been recovered. Currently, the isolates are being identified to the species level. To support the assumption that S. chromogenes is a more host-adapted species and can cause persistent infections, strain typing will be performed. Risk factors for IMI with the more pathogenic CNS species will be analyzed using a mixed logistic regression model with random effects for herd, cow and quarter. 15

16 Clinical mastitis incidence on Flemish dairy farms - preliminary results Joren Verbeke, Sarne De Vliegher M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium Clinical mastitis cases are rarely sampled or recorded on Flemish dairy herds. Consequently, little is known about the incidence and pathogen distribution. Currently, a survey is ongoing to enlarge our knowledge on this important disease. Preliminary results are presented in this abstract. Materials and methods Sixty-seven Flemish dairy herds were randomly selected and offered to participate in the study. All participating herds (n = 52) were visited in September or October Details about the study were explained, sampling material was handed over and a small questionnaire was performed. Farmers were asked to sample each case of clinical mastitis during a period of one year. Milk samples are transported to MCC Flanders (Lier) where bacteriological culture and antimicrobial susceptibility testing is performed. Results are sent to the farmers and herd veterinarians and saved in a dataset. Results and Discussion Fifty-two herds were enrolled in the study indicating a response rate of 78% was achieved. The average herd size is 58 lactating cows (16-240). In total, more than 3000 cows are being followed exceeding the calculated sample size to estimate the proportion of cows with clinical mastitis ±2% with a 95% confidence (n = (Z α 2 pq)/ L 2 = (1.96²*0.46*0.54)/ = 2386). Thirty-three herds are enrolled in the DHI program of the Flemish Cattle Breeding Association. Cows are milked in a conventional milking parlor (n = 36), a rotary milking parlor (n = 3), an AMS (n = 3) or a tie stall (n = 10). Blanket dry cow therapy is applied in 47 herds. Currently, we estimate the incidence of clinical mastitis in Flemish dairy herds at 38.2 quarter cases per 100 cow years at risk. Differences between herds are considerably large (see graph below). Currently, 459 milk samples have been analyzed. In most cases (60%), flocks in milk were the only visible symptom. In 32% of the cases a hard quarter was noticed. Symptoms of systemic illness (e.g. fever) were limited to 8% of the cases. Streptococcus uberis (23%), Escherichia coli (18%), Streptococcus dysgalactiae (9%) and Staphylococcus aureus (8%) were most commonly isolated. A negative result was detected in 19% of the milk samples. In 6% of the cases, three or more bacterial species were cultured indicating that only few samples were contaminated. Conclusion Preliminary results suggest that; - The incidence of clinical mastitis is high on Flemish dairy herds - Herd differences are large indicating room for improvement when reasons for the variability have been identified - Symptoms are mostly limited to flocks in the milk - Environmental pathogens such as S. uberis and E. coli are the main causes of clinical mastitis Acknowledgement This study is financed by Veepeiler Rund and supported by MCC Vlaanderen. 16