STAPHLOCOCCUS AUREUS MASTITIS: WHAT WE NEED TO KNOW TO CONTROL THEM
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REVIEW
STAPHYLOCOCCUS AUREUS MASTITIS: WHAT WE NEED TO KNOW TO CONTROL THEM
Zecconi, A. Department of Animal Pathology, Hygiene and Health. Università degli Studi di Milano, via celoria 10, 20133 Milano, Italy
INTRODUCTION
Among bacteria causing mastitis, only Streptococcus agalactiae, Staphylococcus aureus, Mycoplasma species, and Corynebacterium bovis are considered as fully contagious. Among these, S.aureus, is currently the most frequently isolated contagious pathogen in subclinical and chronic bovine mastitis worldwide (1). The reasons for the large spread of S.aureus intramammary infections (IMI) worldwide are obviously related to bacteria characteristics, but also to a general misunderstanding of the epidemiology of S.aureus IMI, leading to inefficient control measures. The description of all the factors involved in S.aureus IMI should include many different aspects, and it is not possible to cover all of them in a single review. Therefore, in this paper we reviewed the main information useful to develop control programs for S.aureus IMI under field conditions and it is focused on the following topics: diagnosis, major risk factors, therapy, vaccination and control methods. Bacteria characteristics S.aureus are described “ coagulase positive, β-haemolytic, maltose and mannitol fermenting organisms, forming pigmented colonies” (2). Not all strains of S.aureus have characteristics that are consistent with the previous description. Indeed, some S.aureus are α-haemolytic, β-haemolytic, α+βhaemolytic, δ-haemolytic, non-haemolytic and even coagulase negative (3). To cause mastitis S.aureus initially must gain access to the mammary gland through the teat canal and then has to avoid removal by the flushing of the fluids during the milking process. Therefore, the ability to adhere to the epithelial cells and extracellular matrix (EcM) proteins is instrumental to colonize the gland and develop the pathologic process. The adhesion mechanism of S.aureus is complex and includes multiple proteins able to specifically recognize components of the microbial surface that recognize adhesive matrix molecules (MScRAMM) (4), allowing bacterial anchorage in normal and inflamed tissues (5). Adhesive molecules are pivotal in the diffusion of S.aureus within and among herds, but they are only one of the several virulence factors involved in the pathogenesis of S.aureus infections. It is out of the scope of this review to describe all these virulence factors. However, assessing S.aureus genetic patterns is useful to understand its epidemiological features. Several studies (6-8) indicate that virulence of S.aureus strains differ, an indication that strain typing is important. Similarly, transmissibility within a herd differs by strain type
(9). Thus, the importance of evaluating the combination of S.aureus virulence factors has been recently emphasized both in human and veterinary medicine (8, 10, 11).
DIAGNOSIS
Several factors have been shown to influence accuracy for detecting S.aureus IMI using conventional bacteriological culture: i.e. sample type (quarter or composite), inoculum volume, sampling time and frequency. Hence, knowledge of the limits will guide the final decision on the method to be employed to detect S.aureus IMI at a cow or mammary quarter level. It is generally accepted that there is relationship between bulk tank Scc (somatic cell count) and the proportion of cows with S.aureus in the herd (12). However, there is increasing evidence that a large proportion of cows in well-managed herds could be S.aureus positive, but with low SCC both at cow and bulk tank level (13, 14). This could be the result of recently acquired infections, reduced pro-inflammatory activity of strains or efficient host immune response (8, 15, 16). Under field conditions, composite or quarter milk sampling can be applied. However, reliability of culturing composite milk samples compared with quarter samples for the diagnosis of S.aureus IMI 0.01 ml as inoculum volume showed a sensitivity and specificity respectively of 57.9 and 98% (17, 18). while composite milk sampling could be used for routine testing when the main interest is determining prevalence of infection at cow level, the use of this test in control programs, where the proper identification of infected cows is crucial, should be carefully evaluated. Methods to increase diagnostic accuracy One potential source of false positive results is the presence of irregular shedding of bacteria (19). However, recent studies suggested that increasing the inoculum volume to 0.1 ml will significantly reduce the risk of false negative results (20, 21). These latter observations are supported by the studies on the accuracy of diagnosis based on different inoculum volumes. Direct plating of 0.01 ml of milk from established S.aureus infections failed to detect these organisms in about 10% of the samples, while plating 0.05 resulted in 6% of false negative samples (22). Further studies, using 0.01 ml as inoculum volume (23), found a 94.2% agreement between duplicate quarter milk samples positive for S.aureus. These results confirmed earlier reports and suggested that the single quarter sampling method was capable of identifying 82-94% S.aureus quarter infections (23). 93
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REVIEW To overcome some of the diagnostic problems, “augmented methods” have been also proposed. Among them, freezingthawing and centrifugation are considered the most interesting. Freezing may affect viability of organisms contained in milk samples taken for bacteriologic culture but it could increase the sensitivity of the test breaking the clusters. However, (24) found that the presence of S.aureus in subclinical and clinical milk samples kept frozen at about -20ºc for 4, 6 and 16 weeks did not differ significantly. The centrifugation of milk samples has been proposed to overcome the low-shedding cases (25). The results showed that cultures of the sediment significantly increased the number of positive outcomes, in comparison with conventional methods. influence the odds of having clinical mastitis in early lactation. In an extended study by (29), there was a significantly greater percentage of S.aureus isolated from epidermal and mucosal sites of heifers in high prevalence herds. Overall, heifers colonized with S.aureus in the mammary gland area were 3.4 times as likely to calve with S.aureus IMI, and this was the largest risk factor of disease. Once S.aureus enters into the herd, the major vehicle of diffusion among cows is milking machines and improper milking procedures. Indeed, several studies found that S.aureus on milking unit liners could have the same fingerprint as the isolates causing IMI (30-32). Additionally, S.aureus has been isolated from udder cloths (33), thus implicating their role as a fomite in the spread of S.aureus mastitis. Davidson (34) in large study on sites of colonization and IMI found the udder and teat skin to be an important reservoir associated with S.aureus mastitis. Zadoks et al. (35) would suggest that the teat skin is not a likely reservoir for S.aureus IMI. Such an argument was partially supported by (36), but it is opposite to that reported by (29, 31, 37). Matos et al. (38) suggested that there are several other potential sources of S.aureus in a dairy, as this pathogen could be isolated from bedding and air of the parlour. Flies were not a source of S.aureus in this study, which is in contrast to that reported by (39). Roberson et al. (29) showed that in lowprevalence herds (<3%), bedding, insects, and water did not yield S.aureus, but high-prevalence herds did have some of these samples that were positive. There is not a broad consensus on the role played by external sources in S.aureus IMI, out of milking machine and, potentially, by heifers. Therefore, these two factors are the most important ones we should be considered when we decide to apply a control program.
ANALYTICAL METHODS
Milk culture could be performed either on blood-agar plate or using selective media such as Baird-Parker or a modification of the latter technique. However, the different phenotypes could cause several problems in diagnostic laboratories when selective media are applied. In our laboratory for an internal comparison, 50 confirmed S.aureus strains were tested on 3 different media: (blood-agar, Baird-Parker -BP- and Baird Parker supplemented with rabbit plasma and fibrinogen-BPRPF). The results showed that 31/50 hadn’t any surrounding clear zone on BP, 17/50 were coagulase negative on BPR-PF and 10/50 were only weakly positive in this latter medium. Therefore, milk culture on 5% blood agar plate still is the recommended method for S.aureus IMI diagnosis, followed by confirmation of the suspected colonies by other methods such as coagulase-test and biochemical tests. When selective media are applied, it is also recommended to confirm all the suspected colonies by other methods. Independently from the method applied, identifying a single colony of S.aureus is enough to define the quarter (cow) as infected. Very recently, methods based on Real Time PCR have been proposed to improve mastitis diagnosis, including for S.aureus (26). However, data available until now are not sufficient to evaluate if the molecular approach will be useful to improve the accuracy of current diagnostic methods for S.aureus IMI.
Traditionally the prevalence of mastitis in heifers prepartum has been overlooked given the concept that heifers without a developed gland are not susceptible to IMI. Fox et al. (27) reviewed heifer mastitis, estimating prevalence between 1% and 10% of all heifers with S.aureus IMI at parturition. Therefore, heifers could be important risk factor acting both as a reservoir and as a vehicle for S.aureus. waage et al.(28) found that S.aureus was the major cause of clinical mastitis in heifers, with clinical mastitis noted during the first 2 weeks of lactation. whether the heifer was raised on the farm or was imported into the herd did not 94
RISK FACTORS
In practice, the common opinion that S.aureus has a relatively poor cure rate is based more on clinical impressions than on scientific evidences. Indeed, the cure rate for lactation therapy reported in literature has a very broad range (4-92%), a narrower, but still broad range has been reported also for dry-cow therapy (40). The recent debate on the increase in microbial resistance observed in human medicine, and particularly of methicillin-resistant S.aureus strains, increased the concerns on the use of antimicrobials for dairy cow therapy, particularly at drying-off. The discussion is still open, even if there are increasing evidences that the antibiotic therapy for mastitis and dry-cow treatment is not associated either with the development of methicillin-resistant strains or an increase of resistance (41). Despite this evidence, the application of antibiotic therapy following the principles of a prudent use of antibiotics, as proposed by various scientific organizations, should be recommended. To apply a prudent and efficacious protocol for mastitis treatment, different factors should be considered (42): type of pathogen involved, antibiotic Volume 65 (3) 2010
THERAPY
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ISRAEL JOURNAL OF VETERINARY MEDICINE susceptibility patterns, severity of inflammatory response, duration of infection, stage of lactation and age of the cow. Probably the most comprehensive analysis of the different factors affecting cure rate for clinical, subclinical mastitis and dry-cow therapy for S.aureus has been proposed by Sol and co-workers (43-45). Among the different factors, Scc showed to influence S.aureus cure rate after treatment of clinical and subclinical mastitis and at drying-off with lower cure rate when SCC were > 106/ml. Hind quarters showed a lower cure rate (subclinical and drying-off therapy), age and number of infected quarters showed to be significant only for drying-off therapy, with a decrease of the cure rate as age and number of infected quarters increased. when the factors affecting cure rate are considered (i.e. age, Scc), the therapy is applied selecting molecules after susceptibility test and with a rational and efficient protocol, the cure rate could be higher than 75 % both for dry cow-therapy and for treatment of cows after calving (43-45). clinical mastitis in the herd. Results showed that prevalence of S.aureus in quarter milk samples taken at calving and three to four weeks post partum did not differ significantly between the vaccine and control group. Regarding the development of clinical mastitis during the first three months after calving and the prevalence of S.aureus in quarter milk samples taken before the onset of treatment, there were no significant differences between the groups. The SCC was lower in vaccinated than in control heifers. However, the difference was only significant on the third milk test day. Cow Vaccination In dairy cows, one of the first vaccines developed was a heat-killed capsular type A and B S. aureus strains and capsular polysaccharide, and it was used in two herds. The results showed that the vaccinated animals had a decrease in mastitis incidence and higher milk yield in comparison with unvaccinated herd-mates (51). A vaccine containing whole inactivated bacteria with pseudocapsule and α-β toxoid was used in a field trial in Norway (52). The study showed that a significant increase in antibody was observed only for pseudocapsule and α toxoid. Moreover, vaccinated heifers showed a prevalence of S.aureus mastitis of 8.6%, while in control heifers it was of 16%. One of the largest field trials on S.aureus vaccine efficacy was reported by (53). Results showed no overall reduction in the incidence of clinical mastitis, even though differences were observed within individual herds. Overall, the known commercially available S.aureus vaccines have shown limited efficacy under field conditions. A vaccine which would prevent the occurrence of bovine mastitis caused by S.aureus or a vaccine that would augment S.aureus control would be of benefit. More recently, two vaccines were commercially available in some countries, but only for one of these two data are available (54-56). This patented vaccine is derived from 3 field strains of S.aureus which contain a broad spectrum of antigenic and immunogenic properties. The composition enables the vaccine to induce a strong homologous as well as heterologous immune response. The results of the field trials showed a 70% specific protection from infection and almost complete protection from udder inflammation expressed by the low SCC (100x103 cell/ml) of the vaccinated cows. The effect of vaccination on subclinical udder infection revealed a significant clearance of S.aureus udder infection in comparison to the control, which was expressed also by a reduction in SCC to normal range. Because of low rate of spontaneous infection among the heifers in the field trial, no specific protection could be evaluated. However, a significant difference in Scc between the vaccinated and nonvaccinated heifers was found (108x103 and 178x103 cell/ml respectively). A significant difference in milk production (0.5 Kg per cow per day) was found as well. Innovative vaccines developed by the means of molecular methods have been investigated by several research groups (57-60). Results are very encouraging, but none of these vaccines is available in dairy field. Thus, even if S.aureus vaccine was the object of a large 95
VACCINATION
Much interest has been devoted to the development of a vaccine against bovine mastitis caused by S.aureus, but to date the results have not been fully convincing. Indeed, vaccination against S.aureus mastitis must take into account a large number of possible antigens and related interactions with the immune system. These different potential targets for an immune response are reviewed by (46) and these targets include capsules, adhesions, surface proteins and toxins. Early vaccine formulations were composed of microorganisms that were cultured in vitro, killed, and injected systemically with or without toxoids and immunologic adjuvants as reviewed by (47, 48). Several such formulations were shown to increase the spontaneous cure rates of S.aureus IMI as well as to lessen the severity of infection but did not prevent new cases of mastitis, but none of them was broadly and consistently applied in dairy herds.
Heifer Vaccination Nickerson et.al. (49) evaluated a polyvalent S.aureus vaccine in heifers beginning at 6 months of age (with periodic 6-month boosters) to determine if vaccination reduced prevalence of S.aureus mastitis during pregnancy and at calving. Results demonstrated that the percentage of new S.aureus infections during pregnancy was lower in vaccinates than controls (14.3 vs. 25.9%), the percentage of quarters showing chronic S.aureus infection was lower in vaccinates than controls (10.7 vs. 18.8%), and at freshening, the percentage of quarters infected, with S.aureus was lower in vaccinates than controls (8.9 vs. 16.1%). The data demonstrated a positive effect of vaccination in increasing antistaphylococcal antibody titers and in preventing new S.aureus infections when the program was initiated at an early age in heifers raised in a herd with high exposure to this mastitis pathogen. Edinger et al. (50) developed a herd-specific vaccine based on two strains of S.aureus previously isolated from cases of Volume 65 (3) 2010
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REVIEW number of studies, still a vaccine with proven efficacy in commercial dairy herds and in different areas is not available. mammary quarters of all cows are treated at drying-off with a commercial antimicrobial treatment. choice of the product is based on the susceptibility of the S.aureus strains isolated in the herd as determined by use of the disc diffusion method. 5. Treatment of infected lactating cows without clinical signs is restricted to those with ≤ 3 lactations and in their first 30 days of lactation, to avoid treatments with a poor cure rate. These cows are moved to a hospital pen to be sampled 5-7 and 10-14 days after the end of the antimicrobial withdrawal period. Only cows that are judged cured because of negative results of 2 consecutive bacteriologic cultures of milk samples are allowed to leave the hospital pen. 6. Antimicrobial and anti-inflammatory treatments are administered to cows that developed clinical mastitis, independently of their S.aureus infection status, and these cows are moved to the hospital pen to be sampled 7 and 14 days after the end of the withdrawal period. Only cows that were judged cured are allowed to leave the hospital pen to come back to the group of origin. 7. At the herd level, farm managers are advised to improve and keep proper bedding hygiene. 8. The control program is monitored directly by means of monthly visits by a trained practitioner and discussion of analysis results, and indirectly by a weekly check of samples sent to the laboratory, to ensure compliance. Results of this field study suggested that a very low incidence rate can be achieved after about 10 months of the control program. Young cows and freshening cows are the most likely to develop new IMI among uninfected cows when segregation of infected cattle is used; uninfected cattle should be carefully checked and specific procedures should be applied to reduced the risk of infection. It could be suggested that heifers should be housed separately from older cows for at least 2 weeks before calving, to reduce the risk of becoming infected from older cows during periparturient period. The proposed control/eradication program based on segregation showed, on average, to reduce the incidence rate of S.aureus IMI below 2% in 10 months and <1% in 18 months. cost/benefit analysis showed that a positive economic return could be achieved even when starting prevalence of S.aureus IMI are in the range 10-20% (66).
CONTROL PROGRAM
Environmental sources and non-dairy animals do not appear to be significant reservoirs and vectors for the disease. Therefore, a control/eradication program for this pathogen could be hypothesized. The frequency of infected herds and cows, and the high cost of the disease represent the reason to develop control programs. Moreover, the availability of accurate diagnostic procedures and efficacious therapeutical protocols allow for their implementation. To develop a strategy to control the infection, the approaches currently applied are the test-and-cull strategy and control programs based on segregation. culling is often suggested as the only way to control S.aureus (61, 62). However, there is poor scientific evidence either on the efficacy or on the economic return of this approach. Indeed, (63) showed that at the end of a program based on test-and-cull strategies applied in three herds, all the three herds had a very similar culling rate, but only the third one, applying a well-managed program in addition, achieved the control of S.aureus IMI. Therefore, culling could be a component of a control program, mainly as the most efficient way to remove the chronic S.aureus cows. However, as a main method of control it showed to be poorly efficient and with a negative economic impact. control programs based on segregation followed the general principles of contagious mastitis control (64) and was based on precise diagnostic procedures and strict control of segregation of infected cows. we reported the epidemiologic pattern IMI in 9 commercial dairy herds after establishment of a standardized and detailed mastitis control program (65). The main steps in the control program are (Figure 1): 1. Application of a precise and consistent milking procedure that included use of a single-service towel to clean the teat, forestripping, and use of postmilking teat disinfectants of known efficacy. 2. Establishment of a milking sequence to reduce infection risk, by milking healthy cows first, then cows and heifers that had recently entered the herd either through purchase or freshening, and then milking cows with S.aureus IMI last. 3. After the first sampling of all lactating cows at the time of enrolment to segregate infected cows, a precise sampling schedule is used. Purchased cows are sampled 5-7 and 1014 days after entry into the herd, and cows that had recently calved are sampled 5-7 and 10-14 days after calving. cows with S.aureus IMI are segregated and are not sampled again until they have calved. Non-infected cows are sampled again 2, 4, 7, 10, 14 and 18 months after the first sampling. 4. Diagnosis of S.aureus IMI is performed with mammary quarter milk samples by bacteriologic culture on 5% bloodagar media. Quarter samples are collected to increase the sensitivity of detection. Recovery of a single colony of S.aureus is considered a positive result indicating an IMI. All
Programs based on segregation has been applied, but on a smaller scale when compared to Str.agalactiae. Diagnostic problems, poor cure rate, and unknown sources of infection are often the arguments advocated to refute the application of a control program. Moreover, the presence of intramammary infections, without large increase of Scc induces many practitioner and farmers to underestimate the impact on milk quality and yield. However, based on the available scientific and practical evidences we can reasonably affirm that: • S.aureus has a variable, but large economic impact on Volume 65 (3) 2010
CONCLUSIONS
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ISRAEL JOURNAL OF VETERINARY MEDICINE dairy herd; • Diagnosis of S.aureus IMI is feasible with conventional methods by experienced laboratories; • Well-designed S.aureus therapy protocols have a curerate not inferior to the one observed against other intramammary pathogens; • S.aureus is not an obligate parasite of the mammary gland, but the importance of potential reservoirs on other body sites or in the environment significantly decreases as the prevalence of IMI decreases in the herd. Therefore, they will not affect the control programs. Therefore, when control programs are based on few important points such as the isolation or removal of reservoirs, the avoidance of S.aureus transmission during milking, a careful monitoring by a trained practitioner, they will be successful with positive economic returns for both the farmer and the practitioner.
12. Gonzalez, R.N., Jasper, D.E., Farver, T.B., Bushnell, R.B., Franti, c.E. Prevalence of udder infections and mastitis in 50 california dairy herds, In: JAvMA, pp. 323-328 1988. 13. Zecconi, A., Piccinini, R. Intramammary infections: epidemiology and diagnosis. In: XXII World Buiatric Congress - Recent developments and perspectives in bovine medicine, Hannover 18-23/08/2002, pp. 346-359, 2002. 14. Zecconi, A., Piccinini, R., Fox, L.K. Epidemiologic study of intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. JAvMA 223, 684-688.2003. 15. Zecconi, A., Binda, E., Borromeo, V., Piccinini, R. Relationship between some Staphylococcus aureus pathogenic factors and growth rates or somatic cell counts. J. Dairy Res. 72, 203208.2005. 16. Middleton, J.R., Fox, L.K., Gay, J.M., Tyler, J.w., Besser, T.E. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-beta-Dglucosaminidase activity in cattle from eight dairies. J. Dairy Sci. 85, 1133-1140.2002. 17. Morselt, M.L., Lam, T., vanwuijckhuise, L.A., Franken, P., Hartman, E.G., Schukken, Y.H. The reliability of bacteriological culturing of composite milk samples in the diagnosis of sublinical udeder infections in dairy cattle. Tijdschr. Diergeneeskd. 120, 426-430.1995. 18. Lam, T., vanwuijckhuise, L.A., Franken, P., Morselt, M.L., Hartman, E.G., Schukken, Y.H. Use of composite milk samples for diagnosis of Staphylococcus aureus mastitis in dairy cattle. JAvMA 208, 1705-&.1996. 19. Sears, P.M., Smith, B.S., English, P.B., Herrer, P.S., R.N., G. Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J. Dairy Sci. 73, 2785-2789.1990. 20. walker, J., Rajala-Schultz, P.J., DeGraves, F.J. The Effects of Inoculum volume and Extended Sampling on the Microbiological Detection of Naturally Occurring Staphylococcus aureus Intramammary Infections In: 49th NMc Annual Meeting, Albuqyerque NM, pp. 230-231, 2010. 21. Torres, A.H., Rajala-Schultz, P.J., DeGraves, F.J. Diagnosis of intramammary infections at dry-off based on sampling strategy, epidemiology of pathogens, and agreement beyond chance. J. Vet. Diagn. Investig. 21, 427-436.2009. 22. Neave, F.K. Diagnosis of mastitis by bacteriological methods alone. In: IDF Seminar on the control of Mastitis, Reading (710/4/1975), pp. 19-36, 1975. 23. Erskine, R.J., Eberhart, R.J., Hutchinson, L.J., Spencer, S.B., campbell, M.A. Incidence and types of clinical mastitis in dairy herds with high and low somatic cell counts. JAvMA 192, 761765.1988. 24. Schukken, Y.H., Smit, J.A.H., Grommers, F.J., vandegeer, D., Brand, A. Effect of Freezing on Bacteriologic culturing of Mastitis Milk Samples. J. Dairy Sci. 72, 1900-1906.1989. 25. Zecconi, A., Piccinini, R., Zepponi, A., Ruffo, G. Recovery of Staphylococcus aureus from centrifuged quarter milk samples. J. Dairy Sci. 80, 3058-3063.1997. 26. Koskinen, M.T., Holopainen, J., Pyorala, S., Bredbacka, P., Pitkala, A., Barkema, H.w., Bexiga, R., Roberson, J., Solverod, L., Piccinini, R., Kelton, D., Lehmusto, H., Niskala, S., Salmikivi, L. Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. J. Dairy Sci. 92, 952-959.2009. 27. Fox, L.K., chester, S.T., Hallberg, J.w., Nickerson, S.c.,
Zecconi, A. Contagious mastitis control program: the Staphylococcus aureus case. Cattle Practice 14, 67-76.2006. 2. carter, G.R., cole, J.R. Diagnostic procedures in veterinary bacteriology and mycology, 5th Edition. Academic Press, London, 1990. 3. Fox, L.K., Besser, T.E., Jackson, S.M. Evaluation of a coagulase-negative variant of Staphylococcus aureus as a cause of intramammary infections in a herd of dairy cattle. JAvMA 209, 1143-1146.1996. 4. Patti, J., Bremell, T., Krajewska-Pietrasik, D., Abdelnour, A., Tarkowski, A., Ryden, c., Hook, M. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect. Immun. 62, 152-161.1994. 5. Foster, T., Hook, M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6, 484-488.1998. 6. Younis, A., Krifucks, O., Fleminger, G., Heller, E.D., Gollop, N., Saran, A., Leitner, G. Staphylococcus aureus leucocidin, a virulence factor in bovine mastitis. J. Dairy. Res. 72, 188194.2005. 7. Younis, A., Krifucks, O., Heller, E., Samra, Z., Glickman, A., Saran, A., Leitner, G. Staphylococcus aureus exosecretions and bovine mastitis. J. vet. Med. B Infect. Dis. vet. Public Health 50, 1-7.2003. 8. Zecconi, A., cesaris, L., Liandris, E., Daprà, v., Piccinini, R. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microbial Pathogenesis 40, 177-183.2006. 9. Sommerhauser, J., Kloppert, D., wolter, w., Zschock, M., Sobiraj, A., Failing, K. The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme. vet. Microbiol. 96, 91-102.2003. 10. Peacock, S.J., Moore, c.E., Justice, A., Kantzanou, M., Story, L., Mackie, K., O’Neill, G., Day, N.P.J. virulent combinations of Adhesin and Toxin Genes in Natural Populations of Staphylococcus aureus. Infect. Immun. 70, 4987-4996.2002. 11. von Eiff, c., Friedrich, A.w., Peters, G., Becker, K. Prevalence of genes encoding for members of the staphylococcal leukotoxin family among clinical isolates of Staphylococcus aureus. Diagn. Microbiol. Inf. Dis. 49, 157-162.2004.
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Utrech University, Utrecht NL. Fox, K.L. Recovery of mastitis pathogens from udder cloths following several laundering methods. Dairy Food and Environmental Sanitation 17, 14-19.1997. Davidson, I. Observation on the pathogenic staphylococci in a dairy herd during a period of six years. Res vet. Sci. 2, 2240.1961. Zadoks, R.N., van Leeuwen, w.B., Kreft, D., Fox, K.L., Barkema, H.w., Schukken, Y.H., Belkum, A. comparison of Staphylococcus aureus isolates from bovine and human skin, milk equipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. J. clin. Microbiol. 40, 3894-3902.2002. Piccinini, R., cesaris, L., Daprà, v., Borromeo, v., Picozzi, c., Secchi, c., Zecconi, A. The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections. J.Dairy Res. 76, 36-41.2008. Larsen, H.D., Sloth, K.H., Elsberg, c., Enevoldsen, c., Pedersen, L.H., Eriksen, N.H.R., Aarestrup, F.M., Jensen, N.E. The dynamics of Staphylococcus aureus intramamammary infections in nine Danish dairy herds. vet. Microbiol. 71, 89-101.2000. Matos, J.S., white, D.G., Harmon, R.J., Langlois, B.E. Staphylococcus aureus from sites other than the lactating mammary gland. J Dairy Sci 74, 1544-1549.1991. Owens, w.E., Nickerson, S.c. Role of horn flies (Haematobia irritans) in Staphylococcus aureus-induced mastitis in dairy heifers. Am. J. vet. Res. 59, 1122-1124.1998. Leslie, K.E., Dingwell, R.T. Background to dry cow therapy: what, where, why - is it still relevant ? In: NMc Annual Meeting Fort worth (Tx) 26-29/01/03, pp. 5-17, 2003. Erskine, R.J., cullor, J., Schaellibaum, M., Yancey, B., Zecconi, A. Bovine mastitis pathogens and trends in resistance to antibacterial drugs. In: 43rd NMc annual meeting, charlotte (1-4/02/2004), pp. 400-414, 2004. Radostits, S.M. Herd Health: food animal production medicine, 3rd Edition. W.B.Saunders Co., Philadelphia, 2001. Sol, J., Sampimon, O.c., Barkema, H.w., Schukken, Y. Factors associated with cure after therapy of clinical mastitis caused by Staphylococcus aureus. J. Dairy. Sci. 83, 278-284.2000. Sol, J., Sampimon, O.c., Snoep, J.J., Schukken, Y. Factors associated with bacteriological cure during lactation after therapy for subclinical mastitis caused by Staphylococcus aureus. J. Dairy. Sci. 80, 2803-2808.1997. Sol, J., Sampimon, O.c., Snoep, J.J., Schukken, Y.H. Factors associated with bacteriological cure after dry cow treatment of subclinical staphylococcal mastitis with antibiotics. J. Dairy. Sci. 77, 75-79.1994. Foster, T. Potential for vaccination against infections caused by Staphylococcus aureus. Vaccine 9, 221-227.1991. Zecconi, A., Smith, K.L. Ruminant Mammary Gland Immunity. FIL-IDF, Bruxelles, 2003. Middleton, J.R., Luby, c.D., Adams, D.S. Efficacy of vaccination against staphylococcal mastitis: A review and new data. Vet. Microbiol. 134, 192-198.2009. Nickerson, S.c., Owens, w.E., Boddie, N.T. Efficacy of a Staph.aureus mastitis vaccine in dairy heifers. In: International Symposium on Immunology of Ruminant Mammary Gland, Stresa IT 11-14 June 2000, pp. 426-431, 2000. Edinger, D., Tenhagen, B.A., Baumgartner, B., Heuwieser, w. Efficacy of a herd vaccine against Staphylococcus aureus in dairy heifers. In: International Symposium on Immunology of Ruminant Mammary Gland, Stresa IT 11-14 June 2000, pp. 410417, 2000. Yoshida, K., Ichiman, Y., Narikawa, S., Evans, w. Staphylococcal capsular vaccine for preventing mastitis in two herds in Georgia. J. Dairy Sci. 1984. 67, 3, 620-627.1984. Nordhaug, M., Nesse, L., Norcross, N., Gudding, R. A field trial with an experimental vaccine against Staphylococcus aureus mastitis in cattle. 1. clinical parameters. J. Dairy Sci. 77, 12671275.1994. watson, D., Mccoll, M., Davies, H. Field trial of a staphylococcal mastitis vaccine in dairy herds: clinical, subclinical and microbiological assessments. Australian vet. Journal 1996. 74, 6, 447-450.1996. Leitner, G., Yadlin, N., Lubashevsy, E., Ezra, E., Glickman, A., chaffer, M., winkler, M., Saran, A., Trainin, Z. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows. II. Field trial. Vet. Immunol. Immunopathol. 93, 153-158.2003. Leitner, G., Lubashevsky, E., Trainin, Z. Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model. vet. Immunol. Immunopathol. 93, 159-167.2003. Leitner, G., Lubashevsky, E., Glickman, A., winkler, M., Saran, A., Trainin, Z. Development of a Staphylococcus aureus vaccine against mastitis in dairy cows I. Challenge trials. Vet. Immunol. Immunopathol. 93, 31-38.2003. Kerro-Dego, O., Prysliak, T., Potter, A.A., Perez-casal, J. DNAprotein immunization against the GapB and Gapc proteins of a mastitis isolate of Staphylococcus aureus. Vet. Immunol. Immunopathol. 113, 125-138.2006. Shkreta, L., Talbot, B.G., Diarra, M.S., Lacasse, P. Immune responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows. Vaccine 23, 114-126.2004. Yin, R.L., Li, c., Yang, Z.T., Zhang, Y.J., Bai, w.L., Li, x., Yin, R.H., Liu, H., Liu, S., Yang, Q., cao, Y.G., Zhang, N.S. construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18. Vet. Immunol. Immunopathol. 132, 270-274.2009. castagliuolo, I., Piccinini, R., Beggiao, E., Palù, G., Mengoli, c., Ditadi, F., vicenzoni, G., Zecconi, A. Mucosal genetic immunization against four adhesins protects against Staphylococcus aureus-induced mastitis. Vaccine 24, 4393-4402 2006. Saperstein, G., Hinckley, L.S., Post, J.E. Taking the team approach to solving Staphylococcal mastitis infection. vet. Med. 83, 939-947.1988.
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62. Stott, A.w., Jones, G.M., Gunn, G.J. chase-Topping, M., Humphry, R.w., Richardson, H., Logue, D.N., Optimum replacement policies for the control of subclinical mastitis due to S. aureus in dairy cows. J. Agric. Econ. 53, 627-644.2002. 63. Hoblet, K.H., Miller, G.Y. Use of partial budgeting to determine the economic outcome of Staphylococcus aureus intramammary infection reduction strategies in three Ohio diary herds. JAvMA 199, 714-720.1991. 64. white, G. An attempt to control the spread of staphylococcal mastitis by segregation and culling. Vet.Rec. 77, 13841386.1965. 65. Zecconi, A., Piccinini, R., Fox, K.L. Epidemiologic study of intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. JAvMA 223, 684-688.2003. 66. Zecconi, A., Piccinini, R., Romani, S. Results and cost-benefit analysis of a Staph.aureus control program in commercial dairy herds. In: 2nd International Symp. on Mastitis and Milk Quality, vancouver (13-15 sep 2001), pp. 311-315, 2001.
FIGURE
Figure 1 - Control program scheme
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REVIEW
STAPHYLOCOCCUS AUREUS MASTITIS: WHAT WE NEED TO KNOW TO CONTROL THEM
Zecconi, A. Department of Animal Pathology, Hygiene and Health. Università degli Studi di Milano, via celoria 10, 20133 Milano, Italy
INTRODUCTION
Among bacteria causing mastitis, only Streptococcus agalactiae, Staphylococcus aureus, Mycoplasma species, and Corynebacterium bovis are considered as fully contagious. Among these, S.aureus, is currently the most frequently isolated contagious pathogen in subclinical and chronic bovine mastitis worldwide (1). The reasons for the large spread of S.aureus intramammary infections (IMI) worldwide are obviously related to bacteria characteristics, but also to a general misunderstanding of the epidemiology of S.aureus IMI, leading to inefficient control measures. The description of all the factors involved in S.aureus IMI should include many different aspects, and it is not possible to cover all of them in a single review. Therefore, in this paper we reviewed the main information useful to develop control programs for S.aureus IMI under field conditions and it is focused on the following topics: diagnosis, major risk factors, therapy, vaccination and control methods. Bacteria characteristics S.aureus are described “ coagulase positive, β-haemolytic, maltose and mannitol fermenting organisms, forming pigmented colonies” (2). Not all strains of S.aureus have characteristics that are consistent with the previous description. Indeed, some S.aureus are α-haemolytic, β-haemolytic, α+βhaemolytic, δ-haemolytic, non-haemolytic and even coagulase negative (3). To cause mastitis S.aureus initially must gain access to the mammary gland through the teat canal and then has to avoid removal by the flushing of the fluids during the milking process. Therefore, the ability to adhere to the epithelial cells and extracellular matrix (EcM) proteins is instrumental to colonize the gland and develop the pathologic process. The adhesion mechanism of S.aureus is complex and includes multiple proteins able to specifically recognize components of the microbial surface that recognize adhesive matrix molecules (MScRAMM) (4), allowing bacterial anchorage in normal and inflamed tissues (5). Adhesive molecules are pivotal in the diffusion of S.aureus within and among herds, but they are only one of the several virulence factors involved in the pathogenesis of S.aureus infections. It is out of the scope of this review to describe all these virulence factors. However, assessing S.aureus genetic patterns is useful to understand its epidemiological features. Several studies (6-8) indicate that virulence of S.aureus strains differ, an indication that strain typing is important. Similarly, transmissibility within a herd differs by strain type
(9). Thus, the importance of evaluating the combination of S.aureus virulence factors has been recently emphasized both in human and veterinary medicine (8, 10, 11).
DIAGNOSIS
Several factors have been shown to influence accuracy for detecting S.aureus IMI using conventional bacteriological culture: i.e. sample type (quarter or composite), inoculum volume, sampling time and frequency. Hence, knowledge of the limits will guide the final decision on the method to be employed to detect S.aureus IMI at a cow or mammary quarter level. It is generally accepted that there is relationship between bulk tank Scc (somatic cell count) and the proportion of cows with S.aureus in the herd (12). However, there is increasing evidence that a large proportion of cows in well-managed herds could be S.aureus positive, but with low SCC both at cow and bulk tank level (13, 14). This could be the result of recently acquired infections, reduced pro-inflammatory activity of strains or efficient host immune response (8, 15, 16). Under field conditions, composite or quarter milk sampling can be applied. However, reliability of culturing composite milk samples compared with quarter samples for the diagnosis of S.aureus IMI 0.01 ml as inoculum volume showed a sensitivity and specificity respectively of 57.9 and 98% (17, 18). while composite milk sampling could be used for routine testing when the main interest is determining prevalence of infection at cow level, the use of this test in control programs, where the proper identification of infected cows is crucial, should be carefully evaluated. Methods to increase diagnostic accuracy One potential source of false positive results is the presence of irregular shedding of bacteria (19). However, recent studies suggested that increasing the inoculum volume to 0.1 ml will significantly reduce the risk of false negative results (20, 21). These latter observations are supported by the studies on the accuracy of diagnosis based on different inoculum volumes. Direct plating of 0.01 ml of milk from established S.aureus infections failed to detect these organisms in about 10% of the samples, while plating 0.05 resulted in 6% of false negative samples (22). Further studies, using 0.01 ml as inoculum volume (23), found a 94.2% agreement between duplicate quarter milk samples positive for S.aureus. These results confirmed earlier reports and suggested that the single quarter sampling method was capable of identifying 82-94% S.aureus quarter infections (23). 93
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REVIEW To overcome some of the diagnostic problems, “augmented methods” have been also proposed. Among them, freezingthawing and centrifugation are considered the most interesting. Freezing may affect viability of organisms contained in milk samples taken for bacteriologic culture but it could increase the sensitivity of the test breaking the clusters. However, (24) found that the presence of S.aureus in subclinical and clinical milk samples kept frozen at about -20ºc for 4, 6 and 16 weeks did not differ significantly. The centrifugation of milk samples has been proposed to overcome the low-shedding cases (25). The results showed that cultures of the sediment significantly increased the number of positive outcomes, in comparison with conventional methods. influence the odds of having clinical mastitis in early lactation. In an extended study by (29), there was a significantly greater percentage of S.aureus isolated from epidermal and mucosal sites of heifers in high prevalence herds. Overall, heifers colonized with S.aureus in the mammary gland area were 3.4 times as likely to calve with S.aureus IMI, and this was the largest risk factor of disease. Once S.aureus enters into the herd, the major vehicle of diffusion among cows is milking machines and improper milking procedures. Indeed, several studies found that S.aureus on milking unit liners could have the same fingerprint as the isolates causing IMI (30-32). Additionally, S.aureus has been isolated from udder cloths (33), thus implicating their role as a fomite in the spread of S.aureus mastitis. Davidson (34) in large study on sites of colonization and IMI found the udder and teat skin to be an important reservoir associated with S.aureus mastitis. Zadoks et al. (35) would suggest that the teat skin is not a likely reservoir for S.aureus IMI. Such an argument was partially supported by (36), but it is opposite to that reported by (29, 31, 37). Matos et al. (38) suggested that there are several other potential sources of S.aureus in a dairy, as this pathogen could be isolated from bedding and air of the parlour. Flies were not a source of S.aureus in this study, which is in contrast to that reported by (39). Roberson et al. (29) showed that in lowprevalence herds (<3%), bedding, insects, and water did not yield S.aureus, but high-prevalence herds did have some of these samples that were positive. There is not a broad consensus on the role played by external sources in S.aureus IMI, out of milking machine and, potentially, by heifers. Therefore, these two factors are the most important ones we should be considered when we decide to apply a control program.
ANALYTICAL METHODS
Milk culture could be performed either on blood-agar plate or using selective media such as Baird-Parker or a modification of the latter technique. However, the different phenotypes could cause several problems in diagnostic laboratories when selective media are applied. In our laboratory for an internal comparison, 50 confirmed S.aureus strains were tested on 3 different media: (blood-agar, Baird-Parker -BP- and Baird Parker supplemented with rabbit plasma and fibrinogen-BPRPF). The results showed that 31/50 hadn’t any surrounding clear zone on BP, 17/50 were coagulase negative on BPR-PF and 10/50 were only weakly positive in this latter medium. Therefore, milk culture on 5% blood agar plate still is the recommended method for S.aureus IMI diagnosis, followed by confirmation of the suspected colonies by other methods such as coagulase-test and biochemical tests. When selective media are applied, it is also recommended to confirm all the suspected colonies by other methods. Independently from the method applied, identifying a single colony of S.aureus is enough to define the quarter (cow) as infected. Very recently, methods based on Real Time PCR have been proposed to improve mastitis diagnosis, including for S.aureus (26). However, data available until now are not sufficient to evaluate if the molecular approach will be useful to improve the accuracy of current diagnostic methods for S.aureus IMI.
Traditionally the prevalence of mastitis in heifers prepartum has been overlooked given the concept that heifers without a developed gland are not susceptible to IMI. Fox et al. (27) reviewed heifer mastitis, estimating prevalence between 1% and 10% of all heifers with S.aureus IMI at parturition. Therefore, heifers could be important risk factor acting both as a reservoir and as a vehicle for S.aureus. waage et al.(28) found that S.aureus was the major cause of clinical mastitis in heifers, with clinical mastitis noted during the first 2 weeks of lactation. whether the heifer was raised on the farm or was imported into the herd did not 94
RISK FACTORS
In practice, the common opinion that S.aureus has a relatively poor cure rate is based more on clinical impressions than on scientific evidences. Indeed, the cure rate for lactation therapy reported in literature has a very broad range (4-92%), a narrower, but still broad range has been reported also for dry-cow therapy (40). The recent debate on the increase in microbial resistance observed in human medicine, and particularly of methicillin-resistant S.aureus strains, increased the concerns on the use of antimicrobials for dairy cow therapy, particularly at drying-off. The discussion is still open, even if there are increasing evidences that the antibiotic therapy for mastitis and dry-cow treatment is not associated either with the development of methicillin-resistant strains or an increase of resistance (41). Despite this evidence, the application of antibiotic therapy following the principles of a prudent use of antibiotics, as proposed by various scientific organizations, should be recommended. To apply a prudent and efficacious protocol for mastitis treatment, different factors should be considered (42): type of pathogen involved, antibiotic Volume 65 (3) 2010
THERAPY
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ISRAEL JOURNAL OF VETERINARY MEDICINE susceptibility patterns, severity of inflammatory response, duration of infection, stage of lactation and age of the cow. Probably the most comprehensive analysis of the different factors affecting cure rate for clinical, subclinical mastitis and dry-cow therapy for S.aureus has been proposed by Sol and co-workers (43-45). Among the different factors, Scc showed to influence S.aureus cure rate after treatment of clinical and subclinical mastitis and at drying-off with lower cure rate when SCC were > 106/ml. Hind quarters showed a lower cure rate (subclinical and drying-off therapy), age and number of infected quarters showed to be significant only for drying-off therapy, with a decrease of the cure rate as age and number of infected quarters increased. when the factors affecting cure rate are considered (i.e. age, Scc), the therapy is applied selecting molecules after susceptibility test and with a rational and efficient protocol, the cure rate could be higher than 75 % both for dry cow-therapy and for treatment of cows after calving (43-45). clinical mastitis in the herd. Results showed that prevalence of S.aureus in quarter milk samples taken at calving and three to four weeks post partum did not differ significantly between the vaccine and control group. Regarding the development of clinical mastitis during the first three months after calving and the prevalence of S.aureus in quarter milk samples taken before the onset of treatment, there were no significant differences between the groups. The SCC was lower in vaccinated than in control heifers. However, the difference was only significant on the third milk test day. Cow Vaccination In dairy cows, one of the first vaccines developed was a heat-killed capsular type A and B S. aureus strains and capsular polysaccharide, and it was used in two herds. The results showed that the vaccinated animals had a decrease in mastitis incidence and higher milk yield in comparison with unvaccinated herd-mates (51). A vaccine containing whole inactivated bacteria with pseudocapsule and α-β toxoid was used in a field trial in Norway (52). The study showed that a significant increase in antibody was observed only for pseudocapsule and α toxoid. Moreover, vaccinated heifers showed a prevalence of S.aureus mastitis of 8.6%, while in control heifers it was of 16%. One of the largest field trials on S.aureus vaccine efficacy was reported by (53). Results showed no overall reduction in the incidence of clinical mastitis, even though differences were observed within individual herds. Overall, the known commercially available S.aureus vaccines have shown limited efficacy under field conditions. A vaccine which would prevent the occurrence of bovine mastitis caused by S.aureus or a vaccine that would augment S.aureus control would be of benefit. More recently, two vaccines were commercially available in some countries, but only for one of these two data are available (54-56). This patented vaccine is derived from 3 field strains of S.aureus which contain a broad spectrum of antigenic and immunogenic properties. The composition enables the vaccine to induce a strong homologous as well as heterologous immune response. The results of the field trials showed a 70% specific protection from infection and almost complete protection from udder inflammation expressed by the low SCC (100x103 cell/ml) of the vaccinated cows. The effect of vaccination on subclinical udder infection revealed a significant clearance of S.aureus udder infection in comparison to the control, which was expressed also by a reduction in SCC to normal range. Because of low rate of spontaneous infection among the heifers in the field trial, no specific protection could be evaluated. However, a significant difference in Scc between the vaccinated and nonvaccinated heifers was found (108x103 and 178x103 cell/ml respectively). A significant difference in milk production (0.5 Kg per cow per day) was found as well. Innovative vaccines developed by the means of molecular methods have been investigated by several research groups (57-60). Results are very encouraging, but none of these vaccines is available in dairy field. Thus, even if S.aureus vaccine was the object of a large 95
VACCINATION
Much interest has been devoted to the development of a vaccine against bovine mastitis caused by S.aureus, but to date the results have not been fully convincing. Indeed, vaccination against S.aureus mastitis must take into account a large number of possible antigens and related interactions with the immune system. These different potential targets for an immune response are reviewed by (46) and these targets include capsules, adhesions, surface proteins and toxins. Early vaccine formulations were composed of microorganisms that were cultured in vitro, killed, and injected systemically with or without toxoids and immunologic adjuvants as reviewed by (47, 48). Several such formulations were shown to increase the spontaneous cure rates of S.aureus IMI as well as to lessen the severity of infection but did not prevent new cases of mastitis, but none of them was broadly and consistently applied in dairy herds.
Heifer Vaccination Nickerson et.al. (49) evaluated a polyvalent S.aureus vaccine in heifers beginning at 6 months of age (with periodic 6-month boosters) to determine if vaccination reduced prevalence of S.aureus mastitis during pregnancy and at calving. Results demonstrated that the percentage of new S.aureus infections during pregnancy was lower in vaccinates than controls (14.3 vs. 25.9%), the percentage of quarters showing chronic S.aureus infection was lower in vaccinates than controls (10.7 vs. 18.8%), and at freshening, the percentage of quarters infected, with S.aureus was lower in vaccinates than controls (8.9 vs. 16.1%). The data demonstrated a positive effect of vaccination in increasing antistaphylococcal antibody titers and in preventing new S.aureus infections when the program was initiated at an early age in heifers raised in a herd with high exposure to this mastitis pathogen. Edinger et al. (50) developed a herd-specific vaccine based on two strains of S.aureus previously isolated from cases of Volume 65 (3) 2010
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REVIEW number of studies, still a vaccine with proven efficacy in commercial dairy herds and in different areas is not available. mammary quarters of all cows are treated at drying-off with a commercial antimicrobial treatment. choice of the product is based on the susceptibility of the S.aureus strains isolated in the herd as determined by use of the disc diffusion method. 5. Treatment of infected lactating cows without clinical signs is restricted to those with ≤ 3 lactations and in their first 30 days of lactation, to avoid treatments with a poor cure rate. These cows are moved to a hospital pen to be sampled 5-7 and 10-14 days after the end of the antimicrobial withdrawal period. Only cows that are judged cured because of negative results of 2 consecutive bacteriologic cultures of milk samples are allowed to leave the hospital pen. 6. Antimicrobial and anti-inflammatory treatments are administered to cows that developed clinical mastitis, independently of their S.aureus infection status, and these cows are moved to the hospital pen to be sampled 7 and 14 days after the end of the withdrawal period. Only cows that were judged cured are allowed to leave the hospital pen to come back to the group of origin. 7. At the herd level, farm managers are advised to improve and keep proper bedding hygiene. 8. The control program is monitored directly by means of monthly visits by a trained practitioner and discussion of analysis results, and indirectly by a weekly check of samples sent to the laboratory, to ensure compliance. Results of this field study suggested that a very low incidence rate can be achieved after about 10 months of the control program. Young cows and freshening cows are the most likely to develop new IMI among uninfected cows when segregation of infected cattle is used; uninfected cattle should be carefully checked and specific procedures should be applied to reduced the risk of infection. It could be suggested that heifers should be housed separately from older cows for at least 2 weeks before calving, to reduce the risk of becoming infected from older cows during periparturient period. The proposed control/eradication program based on segregation showed, on average, to reduce the incidence rate of S.aureus IMI below 2% in 10 months and <1% in 18 months. cost/benefit analysis showed that a positive economic return could be achieved even when starting prevalence of S.aureus IMI are in the range 10-20% (66).
CONTROL PROGRAM
Environmental sources and non-dairy animals do not appear to be significant reservoirs and vectors for the disease. Therefore, a control/eradication program for this pathogen could be hypothesized. The frequency of infected herds and cows, and the high cost of the disease represent the reason to develop control programs. Moreover, the availability of accurate diagnostic procedures and efficacious therapeutical protocols allow for their implementation. To develop a strategy to control the infection, the approaches currently applied are the test-and-cull strategy and control programs based on segregation. culling is often suggested as the only way to control S.aureus (61, 62). However, there is poor scientific evidence either on the efficacy or on the economic return of this approach. Indeed, (63) showed that at the end of a program based on test-and-cull strategies applied in three herds, all the three herds had a very similar culling rate, but only the third one, applying a well-managed program in addition, achieved the control of S.aureus IMI. Therefore, culling could be a component of a control program, mainly as the most efficient way to remove the chronic S.aureus cows. However, as a main method of control it showed to be poorly efficient and with a negative economic impact. control programs based on segregation followed the general principles of contagious mastitis control (64) and was based on precise diagnostic procedures and strict control of segregation of infected cows. we reported the epidemiologic pattern IMI in 9 commercial dairy herds after establishment of a standardized and detailed mastitis control program (65). The main steps in the control program are (Figure 1): 1. Application of a precise and consistent milking procedure that included use of a single-service towel to clean the teat, forestripping, and use of postmilking teat disinfectants of known efficacy. 2. Establishment of a milking sequence to reduce infection risk, by milking healthy cows first, then cows and heifers that had recently entered the herd either through purchase or freshening, and then milking cows with S.aureus IMI last. 3. After the first sampling of all lactating cows at the time of enrolment to segregate infected cows, a precise sampling schedule is used. Purchased cows are sampled 5-7 and 1014 days after entry into the herd, and cows that had recently calved are sampled 5-7 and 10-14 days after calving. cows with S.aureus IMI are segregated and are not sampled again until they have calved. Non-infected cows are sampled again 2, 4, 7, 10, 14 and 18 months after the first sampling. 4. Diagnosis of S.aureus IMI is performed with mammary quarter milk samples by bacteriologic culture on 5% bloodagar media. Quarter samples are collected to increase the sensitivity of detection. Recovery of a single colony of S.aureus is considered a positive result indicating an IMI. All
Programs based on segregation has been applied, but on a smaller scale when compared to Str.agalactiae. Diagnostic problems, poor cure rate, and unknown sources of infection are often the arguments advocated to refute the application of a control program. Moreover, the presence of intramammary infections, without large increase of Scc induces many practitioner and farmers to underestimate the impact on milk quality and yield. However, based on the available scientific and practical evidences we can reasonably affirm that: • S.aureus has a variable, but large economic impact on Volume 65 (3) 2010
CONCLUSIONS
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ISRAEL JOURNAL OF VETERINARY MEDICINE dairy herd; • Diagnosis of S.aureus IMI is feasible with conventional methods by experienced laboratories; • Well-designed S.aureus therapy protocols have a curerate not inferior to the one observed against other intramammary pathogens; • S.aureus is not an obligate parasite of the mammary gland, but the importance of potential reservoirs on other body sites or in the environment significantly decreases as the prevalence of IMI decreases in the herd. Therefore, they will not affect the control programs. Therefore, when control programs are based on few important points such as the isolation or removal of reservoirs, the avoidance of S.aureus transmission during milking, a careful monitoring by a trained practitioner, they will be successful with positive economic returns for both the farmer and the practitioner.
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Zecconi, A. Contagious mastitis control program: the Staphylococcus aureus case. Cattle Practice 14, 67-76.2006. 2. carter, G.R., cole, J.R. Diagnostic procedures in veterinary bacteriology and mycology, 5th Edition. Academic Press, London, 1990. 3. Fox, L.K., Besser, T.E., Jackson, S.M. Evaluation of a coagulase-negative variant of Staphylococcus aureus as a cause of intramammary infections in a herd of dairy cattle. JAvMA 209, 1143-1146.1996. 4. Patti, J., Bremell, T., Krajewska-Pietrasik, D., Abdelnour, A., Tarkowski, A., Ryden, c., Hook, M. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect. Immun. 62, 152-161.1994. 5. Foster, T., Hook, M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 6, 484-488.1998. 6. Younis, A., Krifucks, O., Fleminger, G., Heller, E.D., Gollop, N., Saran, A., Leitner, G. Staphylococcus aureus leucocidin, a virulence factor in bovine mastitis. J. Dairy. Res. 72, 188194.2005. 7. Younis, A., Krifucks, O., Heller, E., Samra, Z., Glickman, A., Saran, A., Leitner, G. Staphylococcus aureus exosecretions and bovine mastitis. J. vet. Med. B Infect. Dis. vet. Public Health 50, 1-7.2003. 8. Zecconi, A., cesaris, L., Liandris, E., Daprà, v., Piccinini, R. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microbial Pathogenesis 40, 177-183.2006. 9. Sommerhauser, J., Kloppert, D., wolter, w., Zschock, M., Sobiraj, A., Failing, K. The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme. vet. Microbiol. 96, 91-102.2003. 10. Peacock, S.J., Moore, c.E., Justice, A., Kantzanou, M., Story, L., Mackie, K., O’Neill, G., Day, N.P.J. virulent combinations of Adhesin and Toxin Genes in Natural Populations of Staphylococcus aureus. Infect. Immun. 70, 4987-4996.2002. 11. von Eiff, c., Friedrich, A.w., Peters, G., Becker, K. Prevalence of genes encoding for members of the staphylococcal leukotoxin family among clinical isolates of Staphylococcus aureus. Diagn. Microbiol. Inf. Dis. 49, 157-162.2004.
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62. Stott, A.w., Jones, G.M., Gunn, G.J. chase-Topping, M., Humphry, R.w., Richardson, H., Logue, D.N., Optimum replacement policies for the control of subclinical mastitis due to S. aureus in dairy cows. J. Agric. Econ. 53, 627-644.2002. 63. Hoblet, K.H., Miller, G.Y. Use of partial budgeting to determine the economic outcome of Staphylococcus aureus intramammary infection reduction strategies in three Ohio diary herds. JAvMA 199, 714-720.1991. 64. white, G. An attempt to control the spread of staphylococcal mastitis by segregation and culling. Vet.Rec. 77, 13841386.1965. 65. Zecconi, A., Piccinini, R., Fox, K.L. Epidemiologic study of intramammary infections with Staphylococcus aureus during a control program in nine commercial dairy herds. JAvMA 223, 684-688.2003. 66. Zecconi, A., Piccinini, R., Romani, S. Results and cost-benefit analysis of a Staph.aureus control program in commercial dairy herds. In: 2nd International Symp. on Mastitis and Milk Quality, vancouver (13-15 sep 2001), pp. 311-315, 2001.
FIGURE
Figure 1 - Control program scheme
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