MASTITIS AND MILK CULTURE

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1 This manuscript has been published in the IVIS website with the permission of the congress organizers. To return to the Table of Content click here or go to MASTITIS AND MILK CULTURE Emile Bouchard, Jean-Philippe Roy, Denis Du Tremblay Faculté de médecine vétérinaire, Université de Montréal, Sicotte Saint-Hyacinthe, Québec, Canada 1. INTRODUCTION Producers often react to mastitis problems when the situation is out of control. In contrast to reproductive performance, monitoring of mastitis is not well established. Veterinarians should be proactive in monitoring mastitis and recommending a control program. The objectives of a mastitis control program should be to reduce new intramammary infections (IMI) and duration of infection. To avoid new IMIs, it is crucial to prevent transmission of pathogenic agents to the mammary gland and to decrease if not eliminate the reservoir of agents that cause mastitis. The five-point control program, developed over 40 years ago in the UK, has been widely accepted. The program has since been expanded to include the 10-point NMC Recommended Mastitis Control Program (National Mastitis Council, 2001). It includes such aspects as biosafety and monitoring of udder health status, both of which require milk cultures. Other programs targeted on milk culture have been suggested and reviewed (Leslie, 1994; Britten, 2004). Milk cultures can yield information beyond identification of cows infected with contagious pathogens. This article will present a program to monitor udder health at the level of the cow and of the herd with an emphasis on milk cultures. Udder health and bacteriological results pre- and postcalving for heifers will also be presented. 2. UDDER HEALTH AND CONTAGIOUS PATHOGENS In a 1990 study on randomly selected herds in the province of Quebec, we documented a prevalence of 47% for Streptococcus agalactiae and 93% for Staphylococcus aureus in bulk tank samples (Bouchard, 1996). These levels were higher than we expected. In view of these findings, the recommendation of total dry cow therapy was reiterated and a control program based on milk cultures was implemented to detect and either treat or segregate, and eventually cull, infected cows. Ten years later, in , a study done on 476 farms based on one bulk tank sample yielded prevalences of 13.9% for Streptococcus agalactiae and 83% for Staphylococcus aureus infected herds.

2 With such a high prevalence of contagious pathogens, we decided to emphasize the use of milk cultures in a mastitis control program based on identification of infected cows. Other control measures and monitoring methods were developed. 3. MILK SAMPLING AND RECORDING Veterinarians in Quebec use a herd health software program developed at the Université de Montréal (DSAHR for Dossier Santé Animale - Animal Health Record) (Bouchard, 1997). Twice a year, each veterinary practice sends health data from the herds they follow to the veterinary Faculty to add to a database. The database now includes more than 88,931 milk culture results which represent 8% of the total 1,126,278 calvings included in the database. The software generates a list of cows to sample at each herd visit based on the cow events and previous month s SCC following these criteria: fresh cows (F): sample taken more than three days after calving either by the producer or the veterinarian during a herd visit, purchased cows (P): sample taken on cows added to the herd since the last visit. Ideally, purchased cows are cultured prior to arrival in the herd, cows suspected of new IMI during lactation (L): sample taken on cows with an increase in SCC at the last DHI test when compared to the previous test. The default criteria is a 1.5- fold (50%) increase in actual SCC using the last count; the actual SCC must be above 250,000 cells/ml (Dohoo & Meek, 1982). Cows meeting this criteria may already be identified as infected by a contagious pathogen, in which case no culture is taken. Otherwise, we recommend doing a California Mastitis test (CMT) and selecting the affected quarter(s) for culture or taking a composite sample. cows with mastitis (M): sample from the affected quarter, usually taken by the producer before treating the cow. 4. INTERPRETATION OF BACTERIOLOGICAL RESULTS Fresh milk samples are submitted to the laboratory the same day or frozen for shipment and delayed processing. Milk culture techniques are adapted from NMC recommendations, using a cotton swab for plating on TSA plates (5% sheep blood agar). If a sample proves negative after first plating, enrichment procedures are used. These consist in plating the incubated milk sample (35 o C for 6 hours). Interpreting and recording results are essentials. The practitioner attending the herd is responsible for interpreting results entered in the cow s individual file. It is important to enter details of the culture results in the individual record to allow changing the interpretation if necessary. Each bacteria is identified by a short alphabetical code prefixed by the number of colonies on the plate (* for growth after enrichment). Here are some examples and their interpretation:

3 Cow # Reason for culture Bacteriology results * Interpretation * 9 F +AB; *CN 4 3 F ++SS L +CN; +STC; +SS F +++SS; +EC 10? 88 F ++CS; +SS; *SS; *CS P +SS; +CS 2, 3, 10 o r11? 31 P +SS; *AB; 4 16 M +SD; +AB; 4 List of bacteria codes and the interpretation classes: Bacteria codes AB : Staphylococcus aureus, α ß toxin AC : Aerococcus spp ACB : Acinetobacter spp AP : Arcanobacterium pyogenes BC : Bacillus spp. BS : Staphylococcus aureus, ß toxin CH : Coliform (hemolytic) CN : Coliform CO : Contaminant CS : Corynebacterium spp. EC : E. coli ETB : Enterobacter spp. ETC : Enterococcus HS : Hemophilus somnus KB : Klebsiella spp. LEVU : Yeast, Fungus MB : Mycobacterium spp. MC : Micrococcus spp MY : Mycoplasma spp. NA : Nocardia NG : Negative PAST : Pasteurella PR : Proteus spp. PS : Pseudomonas PT : Prototheca SA : Streptococcus agalactiae SAL : Salmonella spp. SB : Streptococcus bovis SD : Streptococcus dysgalactiae SE : Serratia marcescens SH : Staphylococcus (hemolytic) SHY : Staphylococcus hyicus SM : Streptomyces spp. SS : Staphylococcus spp. ST : Streptococcus spp. SU : Streptococcus uberis Interpretation

4 1 : Bulk tank 2 : No significant growth 3 : Suspect 4 : S. aureus 5 : S. agalactiae 6 : S. aureus + S. agalactiae 7 : Mycoplasma 8 : Coliform 9 : Streptococcus non-ag 10 : Staphylococcus spp. 11 : Corynebacterium spp. 12 : Yeast 13 : Contaminated 14 : Other organisms Interpretation of milk culture results is not an exact science and may change upon the clinician s judgement, the herd situation or the consequences of misclassification. The following guidelines are normally used: presence of one colony of a major contagious pathogen (Staphylococcus aureus, Streptococcus agalactiae and Mycoplasma spp.) should always be identified as positive, results from a composite sample can fit into more than one category, multiple pathogens results, the pathogen causing an infection of longer duration should be considered first, major environmental pathogens can also be considered as potential contaminants, especially in mixed culture. For example, cow #10 should be classified as Staph. spp (10) because we might want to select her for dry cow therapy in a selective treatment protocol; it is therefore less important to classify the cow in the coliform category because no immediate action would be required. 5. MILK PATHOGENS DISTRIBUTION Figures 1 and 2 present the results from 14 herds, all clients of the veterinary school ambulatory clinic that were cultured over a period of 4.5 to 16 years (average = 11.5 years). A total of 12,259 samples were cultured: 81% were taken at freshening; 12% during the lactation following an increase in SCC; 6% for mastitis; and 1% for purchased cows. The most prevalent bacteria at calving are Staphylococcus aureus (10%) and coagulase negative Staph. (CNS) (8.9%). Streptococcus agalactiae is no longer a problem in our herds. Streptococci including Streptococcus uberis and Streptococcus dysgalactiae represent 3.4% of the isolates at calving. More than 25% of the cows have an infection at calving. In cases of mastitis, 59.7 % of the cows were positive. Again, Staphylococcus aureus is the most prevalent bacteria (16.9%), but coliforms (16.9%) are equally important. Streptococci constitute the third group with 12.5% of infections. More than 40% of the mastitis samples were negative.

5 Bacteria cultured from fresh cow samples Corynebacterium sp S. agalactiae Other Yeast 2% 0.4% 1% 1% Staph. sp. 9% Strep. sp. Coliform 1% 3% S. aureus 10% Negative 73% Figure 1. Interpretation of fresh cow samples (n = 9,934) Bacteria cultured from clinical mastitis samples Other Yeast 3% 4% Staph. sp. 6% Strep. sp. 12% Coliform S. aureus 17% 17% Negative 41% Figure 2. Interpretation of samples from cows with mastitis (n = 796) 6. MILK CULTURE RESULTS FOLLOW-UP Milk culturing should be emphasized for solving mastitis problems in dairy herds. Bacteriological results when combined with monthly somatic cell count (SCC) increase sensitivity of detection of infected cows and subclinical mastitis. Control measures (treatment, segregation, culling) can be targeted more precisely with culture results. Producers and veterinarians feel that they can take better culling decisions based on the cow infectious status.

6 Follow-up of transmission during lactation is possible. Early detection of new pathogens in the herd is possible. Because milk cultures are not available on a monthly basis, incidence of IMI (new IMIs) in a herd is often evaluated by using SCC. But for pathogen specific infection rate (incidence or prevalence), milk cultures are necessary. The infection rates can be monitored to evaluate udder health improvement in a herd in addition to clinical mastitis rates (Kelton & Godkin, 2000). We can also show a decreased incidence of IMI for Staphylococcus aureus (Figure 3) and possibly of other pathogens with longer follow-up. 10% 8% 6% 4% 2% 0% Years of follow-up Figure 3. Incidence of IMI with Staphylococcus aureus in 14 herds (970 calvings) during the first 3 years after the program was implemented The changing prevalence of infected cows can also be traced. Figure 4 shows a steady decrease of 4% in the prevalence of Staphylococcus aureus every five years over a 10-year period in three herds from the control program. This also serves to demonstrate that a Staphylococcus aureus problem cannot be solved rapidly. 20% 15% 10% 15.7% 12.2% 8.9% 5% 0% Figure 4. Prevalence of Staphylococcus aureus infection in three herds over a 10-year period It is useful to the producer to see what the herd would look like without the chronically infected Staphylococcus aureus cows. Figure 5 is graph of the monthly SCC of the uninfected cows in Herd and Figure 6 is the same graph showing the Staphylococcus aureus infected cows only. We Year

7 can see that the cows with a normal udder health status have an average SCC below 100,000 cells/ml (Perreault et al. 2004). Monthly Somatic Cell Count july aug. sept. oct. nov. dec. jan. feb. mar. apr. Month (Herd 12530) Figure 5. SCC of cows negative at culture in herd Monthly Somatic Cell Count may june july aug. sept. oct. nov. dec. jan. feb july aug. sept. oct. nov. dec. jan. feb. mar. apr. Month (Herd 12530) Figure 6. SCC of cows positive for Staphylococcus aureus in herd INFECTIONS IN HEIFERS may june july aug. sept. oct. nov. dec. jan. feb. First calf heifers mammary gland infection (Trinidad et al. 1990; Nickerson et al. 1995; Oliver et al. 2004) and their treatments (Middleton et al. 2006) are drawing more attention. At the ambulatory clinic of l Université de Montréal, during a one year period in 2001, 71% of the heifers cultured at calving (F) were negative. During the same period, we found the following pathogens: CNS (10.5%), Staphylococcus aureus (7.9%), Streptococcus non-ag. (3.3%) other major pathogens (5%) and Streptococcus agalactiae (0%). A study on pre-fresh heifers was conducted in 2003 at the Université de Montréal in Saint- Hyacinthe. The objective was to evaluate the effect of Pirlimycin (a treatment used during lactation)

8 when administered to heifers 6 to 12 days preceding their first calving on infection rate, production and SCC as compared to a control group. The infection rates obtained for the control group before and after calving are provided in Table I. Table I. Results from a pre-fresh heifer trial: pre and post-calving culture results from the control (no treatment) group Heifers infected (%) Bacteria Pre-calving (n = 197) Post-calving (n = 181) S. aureus CNS Streptococcus spp Other Gram Total Gram Gram - and yeasts Total IMI Our findings on routine fresh samples and the results on the control group of a clinical trial presented in Table I are similar to those reported in other studies. They demonstrate the importance of paying attention to heifers that represent the future of the herd and a possible reservoir of milk pathogens for the herd. 8. SUMMARY Control programs have been developed to improve milk quality and udder health. However, on a practical standpoint, mastitis evaluation and follow-up are not as well established as reproductive follow-up. A program for the follow-up and the evaluation of mastitis using milk cultures is presented. Culture results related to first-calf heifers are also discussed 9. KEY WORDS Mastitis, bacteriology, udder health. 10. RESUME Des programmes de contrôle ont été établis pour améliorer la qualité du lait et la santé du pis. Toutefois, les méthodes de suivi et d évaluation de la mammite dans un troupeau sont moins bien établies que, par exemple, le suivi de la reproduction. Un programme de suivi de mammite intégrant la culture de lait et des méthodes d évaluation de la mammite sont exposés dans cet article. Des résultats concernant la santé du pis des génisses et des primipares sont également présentés. 11. MOTS CLES Mammite, bactériologie, santé du pis. 12. ZUSAMMENFASSUNG Kontrollprogramme wurden erstellt, um die Milchqualität und die Eutergesundheit zu verbessern. Die Betreuungs- und Beurteilungsmethoden bei Euterentzündungen in einer Herde sind aber weniger etabliert als zum Beispiel die Fortpflanzungsbeobachtung. In diesem Artikel wird ein Mastitisbeobachtungsprogramm bestehend aus Milchkultur und Evaluationsmethoden bei einer Euterentzündung vorgestellt. Einige Resultate betreffend der Eutergesundheit von Rindern und Primiparen werden ebenfalls präsentiert.

9 13. SCHLÜSSELWÖRTER Mastitis, Biologie, Eutergesundheit. 14. RESÚMEN Hay planes de control para mejorar la calidad de la leche y la salud de la ubre, sin embargo, los métodos de seguimiento y de evaluación de la mastitis en la manada son menos fiables que otros planes de verificación, como el de la reproducción. En este artículo, exponemos un plan de control de la mastitis que incluye el cultivo de la leche y la evaluación de la mastitis. También presentamos los resultados sobre la salud de la ubre de las vaquillas y de las vacas primerizas. 15. PALABRAS CLAVES Mastitis, bacteriologia, salud de la ubre. 16. REFERENCES Bouchard E, Guillemette JM et al. Mastitis and its control. The principal cause: infectious bacteria. Prod dlait Québec, 1996; 16: Bouchard E, DuTremblay D et al. A review of the Quebec Dairy Herds Health Improvement (ASTLQ) project. Proc 30 th Annual Congress AABP, 1997; Britten A. Implementing and evaluating fresh cow mastitis programs. Proc 43 rd Annual Meeting Nat Mastitis Council, 2004: Dohoo IR, Meek AH. Somatic cell counts in bovine milk. Can Vet J, 1982; 23:119. Kelton DF, Godkin MA. Mastitis culture programs for dairy herds. NMC Proc 39 th Annual Meeting Nat Mastitis Council, 2000: Leslie K. Herd health - Food Animal Production Medicine, 2 nd Edition. Radostits, Leslie & Fetrow, Eds. W.B. Saunders, publisher. Middleton JR, Timms LL et al. Effect of prepartum intramammary treatment with pirlimycin hydrochloride on prevalence of early first-lactation mastitis in dairy heifers. JAVMA, 2005; 227(12): National Mastitis Council, 2001, National Mastitis Council Recommended Mastitis Control Program. Nickerson SC, Owens WE et al. Mastitis in dairy heifers: initial studies in prevalence and control. J Dairy Sci, 1995; 78: Oliver SP, Gillepsie BE et al. Heifer mastitis: prevalence, risk factors and control strategies. Proc 43 rd Annual Meeting Nat Mastitis Council, 2004: Perrault JY, Laporte A et al. Gestion de la santé du pis: le point de vue d un médecin vétérinaire praticien. Proc Sympo Bovins Laitiers, 2004:2-22. Trinidad PS, Nickerson C et al. Prevalence of intramammary infection and teat canal colonization in unbred and primigravid dairy heifers. J Dairy Sci, 1990; 73:

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