Vaccination Strategies for Mastitis

Transcription

1 Vaccination Strategies for Mastitis R.J. Erskine, DVM, PhD KEYWORDS Dairy Mastitis Vaccination KEY POINTS Prevention of mastitis primarily relies on consistent application of proper milking practices and providing a clean dry environment for the cow. Use of core-antigen Gram-negative bacterins to reduce the severity of clinical coliform mastitis has been demonstrated to be successful, although limitations of this technology exist. Use of bacterins and other products to immunize dairy cattle against mastitis must be integrated with a total herd health management program. Prevention of exposure is the foundation of infectious disease control programs, including mastitis. The tenets of mastitis prevention are maintaining cows in a clean, dry, comfortable environment and ensuring that recommended milking practices are consistently followed. Under the proper circumstances, vaccination can augment a herd mastitis control program. However, vaccination is essentially an insurance policy to mitigate losses that result from exposure, and subsequent infection, from mastitis pathogens. Similar to an insurance policy, the purchaser of the plan has to weigh expected benefits against costs and select a policy that best serves their individual needs. Thus, veterinarians who counsel dairy producers on mastitis vaccination programs should be able to assess the need, evaluate the available vaccines that could help resolve the problem, and establish a program that balances applied immunology with logistical reality of the dairy operation. Vaccination protocols should be designed to meet individual herd needs, and only applied as part of a comprehensive mastitis prevention program, and not as a proxy for inadequate management. This article will (1) briefly review immunologic principles of immunization, (2) consider the principles of evaluating the efficacy of mastitis vaccines, (3) cite the potential benefits and limitations of available commercial products and the most promising research, and (4) propose guidelines for immunization protocols. The author has nothing to disclose. Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, D-202 Veterinary Teaching Hospital, East Lansing, MI 48824, USA address: erskine@cvm.msu.edu Vet Clin Food Anim 28 (2012) vetfood.theclinics.com /12/$ see front matter 2012 Elsevier Inc. All rights reserved.

2 258 Erskine VACCINE IMMUNOLOGY AND EFFICACY Vaccination is a controlled exposure of the host defense system to a pathogen, or toxin, that ideally is robust enough to impart the immune system with increased surveillance and intensity of response, should that particular antigen be encountered again, but without the pathology of an actual infection. For better understanding of immunologic principles, and specifically mammary immunology, the reader is advised to seek in-depth reviews. 1 3 Microbes are the primary cause of bovine mastitis, with the predominance of infections caused by bacteria. If invading pathogens are able to overcome the teat canal barrier, and gain entry into the gland, a series of innate host defenses help to limit bacterial growth. These include soluble factors such as complement, lactoferrin, and immunoglobulin and a cellular response that (1) functions to identify foreign agents, (2) changes local vasculature and recruits immune effectors (inflammation), and (3) enhances pathogen destruction though ingestion and killing by phagocytes. Innate host defenses differentiate host tissue from pathogens by recognition of molecules with regular patterns that are universal to many microbes but not on the body s own cells. 4 These pathogen-associated molecular patterns (PAMPs) are recognized and selectively bound by serum proteins and receptors in host cells, which are termed pathogen recognition receptors (PRRs). 4 Toll-like receptors (TLRs) are an important class of PRRs that reside both on the cell surface and within host cells. These receptors act as the critical link between recognition of foreign agents and initiation of the host response by invoking gene expression and release of inflammatory cytokines (eg, interleukins, tumor necrosis factor- ) from the host cells. 4,5 Examples of specific pathogen targets bound by TLRs include peptidoglycan and lipoteichoic acid of gram-positive bacteria (TLR-2), gramnegative lipopolysaccharide (LPS; TLR-4), bacterial flagellin (TLR-5), and bacterial DNA, as CpG oligonucleotides (TLR-9). 1 Thus, much of both local inflammation and, if the infection is severe enough, systemic signs associated with mastitis are attributable to this response. Numerous cell types, including epithelial and endothelial cells, have been identified as possessing TLRs, which can contribute to an inflammatory response. 2 In addition to release of proinflammatory cytokines, phagocytic cells, such as macrophages, neutrophils, and natural killer cells, actively eliminate pathogens. 1 If a pathogen survives innate host defenses, the adaptive, or acquired, immune system is triggered. This branch of the immune system recognizes specific antigens of pathogens, and if repeated exposure occurs, an immunologic memory initiates a faster and more intense response, of longer duration. It is antigen specificity, and the subsequent amnestic response, of the acquired immune response that serves as the basis for vaccination. The primary cells of acquired immunity are the lymphocytes. To date, the roles of the CD4 (helper), CD8 (cytotoxic and suppressor), and CD17 (phagocyte agonist) subsets in bovine mammary immunity are best understood. 2,6,7 B-lymphocytes, both as potential antigen processors and as precursors to immunoglobulin-producing plasma cells, also play a critical role. Lymphocytes will respond to a specific microbial antigen only if the antigen (protein) is combined with a major histocompatibility complex (MHC) molecule on the surface of host cells, a process referred to as antigen presentation. 2 Cells such as macrophages and dendritic cells are particularly efficient in antigen processing, by phagocytzing, digesting, and presenting antigens on their membrane surface in conjunction with and MHC molecule. In response to antigen presentation, naïve lymphocytes then undergo clonal expansion and differentiation. A specific antigen

3 Vaccination Strategies for Mastitis 259 can only be presented to a lymphocyte that solely recognizes that antigen. 4 Thus, successful immunization against a pathogen must link the invasion of the pathogen with recruitment of innate phagocytic cells, that recognize and digest the microbe, and subsequently present one of potentially hundreds of different protein antigens from the pathogen on the cell surface, in order attract and activate a matching lymphocyte that specifically recognizes the antigen. T-helper cells play a central role by (1) enhancing B-lymphocyte clonal expansion and differentiation to antibodyproducing plasma cells and (2) completing the inflammatory response loop by releasing cytokines such as interleukin (IL)-17 and interferon- that stimulate phagocytes to more efficiently phagocytize and kill ingested microbes. Antigen-specific antibodies that arise from the acquired immune response also enhance phagocyte function by opsonizing microbes, thus assisting in target recognition and ingestion. In ruminants, the lactating mammary gland must overcome numerous deficits relative to other tissues, in order to effectively respond to pathogen invasion. SCC, which in lactating dairy cattle are primarily leukocytes, are typically less than 10 5 cells/ml in uninfected glands, which is approximately 100-fold lower than in blood. Concentrations of factors such as immunoglobulin, lactoferrin, and complement are also lower in milk relative to plasma. 8 Additionally, the ability of circulating macrophages in milk to reenter tissue for antigen presentation remains speculative. Both macrophages and neutrophils in milk have decreased phagocytic ability, relative to blood. 3,9 Furthermore, phagocyte diapedesis and killing and lymphocyte mitogenic responses are markedly decreased during the periparturient period, and this may help contribute to increased incidence of mastitis at this stage of lactation. 7,10 13 Cows infected with Staphylococcus aureus have a subpopulation of CD8 lymphocytes within the mammary gland that suppress proliferation of CD4 lymphocytes, and this class of CD8 cells is preferentially trafficked into the gland during the postpartum period. 6 Host-adapted pathogens, such as S aureus, with numerous virulence factors to help evade recognition and phagocytosis, further compromise the ability of host defenses to eliminate the infection. Thus, vaccination to prevent intramammary infection (IMI) following entry of pathogens into the gland has numerous barriers to overcome relative to other tissues. Considerable variation in response to immunization between animals will exist even under ideal conditions. However, immunizing under such conditions as periods of extreme heat, which is especially pertinent for lactating cows, immediately after transportation, and concurrent outbreaks of other pathogens, such as Salmonella sp or Mycoplasma sp, should be avoided. Recent reports have suggested that cows with marked negative energy balance may also have profound alterations in immune cell function and expression of fatty acid precursors to inflammation. 14 Avoiding vaccination against severe coliform mastitis during stages of lactation when negative energy balance is typical may be problematic; this stage of lactation is also associated with increased risk of clinical mastitis. The management labor culture is also important to optimize vaccine response. With present day marketing of vaccines through a variety of farm supply outlets, ensuring compliance with adequate immunization protocols is a critical need for veterinary oversight on many dairies. In a Pennsylvania study, although 82% of dairy producers indicated that they routinely vaccinated their herds for bovine viral diarrhea, only 27% of the herds were found to be adequately vaccinated. 15 The decision to employ a vaccine as part of a mastitis control program should be founded on the practitioner s ability to access peer-reviewed studies and to compare incidence and severity of natural infections between vaccinated animals and unvaccinated controls. Such studies should encompass a diversity of herd management

4 260 Erskine practices and geographic regions. Unfortunately, such trials require large number of animals and long duration. Alternatively, challenge-exposure trials are often performed to assess vaccine efficacy. This type of trial requires fewer animals and less time, and many variables such as different strains of bacteria, challenge dose, and physiologic and lactation status of the animal can be uniformly standardized. Additionally, more comprehensive outcomes, including shedding of bacteria in milk, physical examination of the cow, and changes in biochemical parameters of serum and milk, can be reliably measured. However, these studies may have limited application in relation to the diversity of dairy farm management and environment, as well as among pathogens. Changes in antibody levels, or titers, often serve as the gold standard to assess the response for an individual animal, or a population, to a vaccine. Failure of a vaccine to elevate titers is often regarded as a vaccination failure. However, predicting direct correlations between antibody titers and clinical outcome in the face of natural exposure is tenuous. A cow with half the serum titer for anti-j5 Escherichia coli antibodies compared to another cow is not twice as likely to succumb to severe coliform mastitis. Likewise, available research often does not report differences in cell mediated responses between vaccinated and unvaccinated cattle, which may be a better indicator of the duration of the amnestic response. Appraisal of vaccine efficacy, as with therapeutics, should not be made on testimonials, especially herd-level impressions of before-and-after responses. Assertions of are often made on perceptions that initiating a vaccine program decreased the incidence and severity of mastitis cases, lowered somatic cell counts (SCC), increased milk production, etc, without employing a concurrent nonvaccinated control group for comparison. This is a frequent flaw in the evaluation of autogenous bacterins, in addition to a lack of quality control standards that are rigorously followed in commercial production. Mastitis incidence and SCC within a herd can rise and fall from many factors, including weather, changes in proportion of younger to older animals in the herd, history of herd additions, correction of milking techniques or deficiencies in equipment, and culling decisions. Practitioners should realistically ask if the goal of immunization is to prevent new IMI or mitigate the severity of infections. The later goal would be prudent in the case of J5 E coli bacterins. Ultimately, a vaccine should be cost effective, present a minimal risk for anaphylaxis and injection lesions, and not affect milk production of lactating cattle. Finally, a vaccine should be administered as part of a herd mastitis control program only after ensuring that the targeted pathogen is a legitimate endemic concern for the udder health of the herd. CORE-ANTIGEN GRAM-NEGATIVE BACTERINS Gram-negative core-antigen bacterins (GNCABs) are commonly used on many dairy herds. The most extensively studied variants are formulated with a mutant strain of E coli O111:B4 (Rc mutant, commonly termed J5) lacking the O antigen capsule of the cell wall but with the core LPS, membrane proteins, and lipid A antigens intact. These core antigens are highly conserved among Gram-negative bacteria and elicit cross-reactive anti Gram-negative antibodies in J5 vaccinated cows Thus, dairy cattle immunized that with these bacterins develop immune resistance against a wide variety of gram-negative bacteria, including mastitis-causing coliforms. Initial studies in California determined that higher serum anti-j5 E coli immunoglobulin G (IgG) was correlated with a lower incidence of clinical coliform mastitis. 21 A subsequent field trial demonstrated that cows administered 3 immunizations at

5 Vaccination Strategies for Mastitis 261 drying off, 30 days after drying off, and in the postpartum period had a 5-fold decrease in the rate of clinical coliform mastitis in the first 100 days of lactation, compared to unvaccinated cows. 22 A field trial in Ohio using a similar dose regimen reported that J5 immunization did not reduce the prevalence of gram-negative IMIs at calving; however, 67% of the infections present at calving in unvaccinated cows developed clinical mastitis, compared to only 20% of the J-5 vaccinated cows. 23 Additionally, vaccination with J5 bacterin increased anti-j5 E coli IgG in serum and milk compared to unvaccinated controls, although anti-j5 E coli antibody titers in milk are orders of magnitude lower than in serum. Following intramammary challenge of E coli, J-5 immunization reduced the severity of infection, but infections were not prevented. 23 The pathogenesis of severe coliform mastitis is dependent on LPS-induced immune mediator responses, and a high proportion of severe coliform mastitis cases become bacteremic. 26 Thus, J5 bacterins may likely have a greater impact on the systemic effects of coliform mastitis, rather than local mammary inflammation. This concept is supported by a field trial from New York that determined 2 doses of J5 bacterin decreased the proportion of clinical mastitis cases that resulted in culling or death by 3-fold but did not reduce the overall incidence of clinical mastitis cases. 27 However, coliform mastitis cases contracted during the first 50 days of lactation resulted in less milk production loss among J5 vaccinated cattle. 28 Gram-negative bacterins are regarded as weakly immunogenic in dairy cattle because they elicit poor amnestic IgG1 and IgG2 responses. 3 An Ontario study found that a substantial population of cows that were administered 2 J-5 bacterin doses during the dry period had poor antibody responses and a higher incidence of mastitis in the subsequent lactation. 29 This relatively short duration of immune protection is supported by several trials A New York study determined that cows vaccinated with 2 doses of J5 bacterin had less milk production loss following clinical mastitis and better survival in the herd compared to unvaccinated cows. However, this protection, as well as anti-j5 antibody titers, declined as lactation (and time since vaccination) progressed and was not existent by 75 days in milk. 30 In a Michigan study, hyperimmunization of cows with 6 doses of J5 bacterin resulted in a 3-fold decrease in severe clinical coliform mastitis from 42 to 126 days in milk, compared to cows that received 3 doses. This suggested that supplemental immunizations extended protection beyond that offered by traditional vaccination regimens. 31 A subsequent Michigan study determined that cows immunized with 5 doses of J5 bacterin had elevated anti-j5 E coli antibody IgG1 and IgG2 titers, relative to unvaccinated cows, for up to 60 to 90 days longer than cows administered 3 doses of J5 bacterin only. 32 Additionally, the best response to immunization, in terms of intensity and duration of antibody response, was gained when multiple immunizations were given as a series in different anatomical locations on the cow. 32 In addition to the J5 E coli bacterin, an Re-17 mutant of Salmonella typhimurium bacterin toxoid is also available commercially. Peer reviewed literature in support of this product is limited. However, in an Arizona study, 2 immunizations in late pregnancy of this product was reported to reduce the incidence of clinical coliform mastitis cases and subsequent culling from coliform mastitis cases, during the first 150 days of lactation. 33 A recent product has been introduced in Europe that combines J5 E coli bacterin with S aureus SP 140 strain. At the time of this writing, evidence of the efficacy of this product to mitigate severe coliform mastitis, in peer reviewed literature, is lacking.

6 262 Erskine Diagnose Does culture of clinical mastitis samples yield coliform organisms frequently? Yes - Determine severity Does severe clinical mastitis occur in > 1 to 2% of the lactations? (especially in > 2 nd lactation cows) NO Re-evaluate the need for coreantigen bacterin immunization Yes- Describe herd epidemiology Days in milk, lactation, season, etc NO Re-evaluate the need for coreantigen bacterin immunization Decrease risks for new infection Bedding, stocking rate, heat stress, transition and other infectious diseases Develop Protocol Number of doses Immunization schedule Target animals Fig. 1. Flow chart to design immunization protocols for dairy cattle with GNCABs. STRATEGIES FOR IMMUNIZATION PROTOCOLS: GRAM-NEGATIVE CORE ANTIGEN BACTERINS Use of GNCABs relies on 5 fundamental principles: (1) diagnose if coliform organisms cause a substantial portion of clinical mastitis within a herd, (2) determine the severity and incidence of clinical coliform mastitis cases, (3) describe the epidemiology of the problem in the herd, (4) decrease other herd risk factors that may contribute to clinical coliform mastitis, and (5) develop a vaccination protocol that best integrates immunology with herd needs and practical limitations. A flow chart to summarize this approach is presented in Fig Diagnose if coliform organisms cause a substantial portion of clinical mastitis. The most practical method to diagnose causative agents of clinical mastitis on dairy farms is milk bacteriology. Although culture of milk samples from individual clinical mastitis cases may often yield negative results, if a representative number of cases are sampled and cultured, the role of coliform organisms as causative agents will be better understood. Diagnosis of causative pathogens for mastitis

7 Vaccination Strategies for Mastitis 263 cases cannot be based on clinical signs, appearance of milk from the affected quarter, or response to antimicrobial therapy. Failure to appreciate the demographics of causative agents in a herd leads to false expectations for a variety of mastitis control measures, including GNCAB use. This disappointment will especially be predictable in herds that experience a majority of clinical mastitis cases caused by water borne organisms such as Pseudomonas sp and Prototheca, mycotic organisms, Mycoplasma bovis, and gram-positive cocci. Typically, in low SCC herds ( 200,000 cells/ml), 30% to 40% of cultured samples will yield coliform organisms, and if samples where no organism was isolated are excluded, the proportion of coliform cases can be 50% to 70%. Nonetheless, this should be confirmed in the laboratory. 2. Determine the severity and incidence of clinical coliform mastitis cases. DeGraves and Fetrow 34 predicted J5 E coli bacterin use to be profitable if clinical coliform mastitis occurs in greater than 1% of lactations and would be profitable at all production levels. However, this estimate may have been conservative. Costs of therapy have increased, and the role of coliform cases in chronic, recurring mastitis has been better elucidated, and in some cases, host-adapted strains may act as a reservoir of infection to other cows. 35 Nonetheless, the primary benefit gained from GNCAB use is the mitigation of the severity, not the prevention of IMI. Ultimately, the frequency of severe clinical mastitis, caused by gram-negative organisms, is the primary motivator to immunize with these bacterins. 3. Describe the epidemiology of the problem in the herd. The consensus of clinical trials and on farm use of GNCABs decreases the incidence of severe clinical mastitis and culling losses among vaccinated cows, in approximately the first 2 to 3 months of lactation. However, a careful review of the distribution of clinical mastitis cases, by stage of lactation, season, and lactation number, should be assessed on an individual herd basis. In some herds, a preponderance of severe mastitis cases may occur after peak milk (3 5 months after calving) and continue well into lactation. 31 Additionally, in many herds, cases are relatively rare in first lactation cows, have seasonal cycles, and may even be associated with routine changes in bedding. It is important to know the when, where, and who of severe mastitis cases in each herd, as this will alter the strategy of immunization regimens. 4. Decrease other herd risk factors that may contribute to clinical coliform mastitis. It is not in the scope of this chapter to outline the complete epidemiology of coliform mastitis. However, primary risk factors, such as clean bedding, udder hygiene, and teat end condition, all play a role. Immunization with GNCABs should only be a part of a herd mastitis prevention program if fundamental mastitis control practices are part of herd routine management. 5. Develop a vaccination protocol that best integrates immunology with herd needs and practical limitations. Ideally, immunization regimens offer a long duration of protection, preferably on an approximately annual cycle relative to calving. Many gram-negative bacterins, not just GNCABs, are limited in terms of duration of protection. Thus, if the response to labeled dosing of a GNCAB is falling short of herd mastitis goals, then additional doses may be considered to better target the occurrence of severe mastitis cases. Although hyperimmunization may be a useful tool, serum anti-j5 antibodies in cows administered 6 doses of J5 bacterin did not differ from cows administered 3 doses, when assayed at the start of the next lactation. 31 Newer vaccines, formulated with adjuvants that activate TLRs of innate immune cells, are under development in human and veterinary medicine. These may offer longer duration of protection and have been investigated for use in J-5 E coli bacterins. 36

8 264 Erskine Care should be exercised in administering multiple gram-negative bacterins (leptospirosis, k-99 E coli, Histophilus somni, etc). LPS contamination is pervasive, although highly variable among gram-negative bacterins. One report determined a transient, 7% decline in milk production following immunization of lactating cows with a J5 bacterin. 37 Veterinarians should inquire of vaccine suppliers, on behalf of their clients, as to the LPS levels in vaccines. If herd records support the strategy, many herds forego the use of GNCABs in first lactation cows or reserve immunization for seasons identified as high risk for severe coliform mastitis. Additionally, there is nothing sacred in dosing GNCABs during the standard dry period/post-calving regimens. Most cows will respond with increased antibody titers at about 10 to 14 days following immunization. If a need is identified to protect cows from severe coliform mastitis during the first 3 weeks after calving, an advantage may be gained by immunizing cows before drying off, at dry off, and 2 weeks before expected calving to maximize antibody titers at the time of calving. Rotation of serial injections to different anatomical injection sites improved the intensity and duration of the antibody response in periparturient dairy cows. 32 However, immunization protocols that are unnecessarily complicated and lead to poor compliance are best avoided. Staphylococcus Aureus S aureus mastitis occurs in many dairy herds. Because of the predominantly contagious nature of this organism, many herds have been able to maintain a low prevalence of IMI caused by this organism. Heifers have been demonstrated to be infected with this pathogen at calving, although this varies greatly between herds and geographic regions. 38 This pathogen generally causes only a small percentage of clinical mastitis cases, and subclinical infections often become chronic and unresponsive to therapy. The ability of this pathogen to establish long-term IMI varies between strains. However numerous virulence factors increase survival of the microbe in host tissues. As the duration of infection increases, fibrin deposition and microabscess formation further reduce the effectiveness of the immune response. The ability to survive intracellularly within phagocytes impairs both humoral immunity and drug therapy. Additionally, S aureus infections rarely elicit marked innate immune responses, compared to E coli, for example. 39 This allows the organism to avoid an acute phase immune response that may endanger existence in infected tissue. 39,40 An effective vaccine against this pathogen needs to overcome several major obstacles: (1) marked variation in strains, thus the need for a conserved, universal antigen, (2) immune stealth virulence factors, especially the ability to survive intracellularly and unexposed to antibodies, and (3) difficulty in assessing impact of a vaccine on reducing deleterious clinical effects of infection and actual IMI status. The last point reflects the nature of S aureus IMI to shed bacteria in milk from infected glands in a cyclic fashion. Recurrence of S aureus in milk may occur up to 28 days after therapy in as much as 80% of quarters because of L-form transformation (an induced variant that lacks cell walls), and rigorous serial sampling should be part of a protocol to determine IMI because of low sensitivity for 1, or even 2 samples, to correctly identify negative quarters. 41,42 Considerable effort, encompassing numerous antigens, virulence factors, and bacterial strains, has been made to develop an efficacious and practical S aureus vaccine. Much of the interest has focused on primiparous heifers. A bacterin containing a lysed culture of polyvalent phage types, including a variety of capsular serotypes, is commercially available in the United States (Lysigin; Boehringer Ingelheim, Ridgefield, CT, USA). This product originated from work in Louisiana that

9 Vaccination Strategies for Mastitis 265 determined 2 initial doses 2 weeks apart, followed by doses at 6-month intervals, reduced IMI at calving caused by both coagulase-negative staphylococci (CNS) and S aureus. 43 However, a subsequent challenge-infection study determined that this bacterin did not prevent IMI, accentuate clearance of IMI, affect SCC, or milk yield post-challenge. 44 The Lysigin-vaccinated heifers did have improved clinical scores and shorter duration of clinical mastitis. 44 Although immunization with Lysigin increased serum anti S aureus IgG1 in heifers, milk concentrations of IgG1, IgG2, or IgM were not affected. 45 In a subsequent report in lactating cows, representing multiple parities, 2 doses of Lysigin administered 14 days apart did not reduce the number of mammary quarters that developed new S aureus or CNS IMI, the time after vaccination to develop new IMI, or SCC. 46 The authors speculated that the bacterin may have induced insufficient opsonizing antibodies in milk to promote phagocytosis. 46 A different formulation of a whole cell killed bacterin was found to reduce the number of quarters infected and SCC following an experimental challenge, although this effect was only reported for up to 13 days after the challenge. 47 A subsequent field study reported that 2 simultaneous doses administered to heifers, followed by an additional dose, resulted in higher anti S aureus specific antibodies throughout the subsequent lactation. The investigators also reported that vaccinated animals had an average increase of 0.5 kg of milk/d and lower SCC. 48 In this study, incomplete Freund s adjuvant was used as part of the initial biphasic dose. This adjuvant is known to cause significant injection site reactions and is not generally used in commercial products. One of the more intriguing developments by the same research group has identified a Target of RNAIII Activating Protein (TRAP), a highly conserved membrane protein among many species of staphylococci, including S aureus. This antigen may have the potential to become a specific and universal anti-staphylococcal vaccine. 49 S aureus produces adhesins, virulence factors that promote attachment to host tissues and subsequent adhesion among bacterial cells, producing a biofilm that can resist phagocytosis. Surface polysaccharides are a key element of staphylococcal biofilm, and strains that express exopolysaccharide (slime associated antigenic complex [SAAC]) in high levels have been isolated. 50 A preliminary study demonstrated the immunization with a high-producing SAAC S aureus strain reduced the concentration of bacteria in infected quarters, compared to a bacterin composed of a low-producing SAAC strain or unvaccinated controls. 50 However, there was no effect on new IMI, and effects of vaccination were followed for only 14 days after challenge. A commercial formulation of the SAAC S aureus bacterin, combined with E coli, has been approved in the European Union. Clinical reports suggest that this product improves udder health by reducing new infection rates and lowering SCC in vaccinated animals. 51,52 However, data presented thus far have not appeared in peer-reviewed publications, and details as to evaluation of new infection rates and cures have been based on tenuous definitions of IMI. Little data have been reported regarding the efficacy of the E coli component of this bacterin. Use of S aureus vaccines would likely have limited use in many dairies, especially in herds with low prevalences of IMI, as is typical for herds with SCC less than 200,000 cells/ml. Thus, it is unlikely that S aureus bacterins will have a significant impact in herds that successfully control contagious mastitis by practicing excellent milking techniques and maintaining milking equipment. Conversely, herds that immunize in lieu of good management practices are likely to have disappointing results. Heifers may provide a viable opportunity for use of an effective staphylococcal bacterin, especially in herds where IMI in post-partum

10 266 Erskine heifers are endemic. As previously mentioned, this varies greatly between herds and geographic regions. If a herd has a staphylococcal mastitis problem, bacterins may also reduce shedding of bacteria from milk of infected animals. Investigators have administered S aureus bacterins in an attempt to augment antimicrobial therapy, with conflicting results. 53,54 Streptococcus sp With the exception of Streptococcus uberis, little progress has been made in the development of vaccines against streptococcal mastitis. Arguably, with the ability to nearly eradicate Streptococcus agalactiae and, to some extent, Streptococcus dysgalactia, with effective control programs, the need for research to develop a vaccine against this group of pathogens is limited. However, control of S uberis has proved more difficult and is an important cause of both clinical and, more typically, subclinical mastitis. IMIs from this pathogen may occur throughout lactation and become chronic, and the pathogen has been demonstrated to internalize and persist in bovine mammary epithelial cells. 55,56 Thus, internalization may aid S uberis to avoid opsonization by antibodies and other innate serum factors, as well as phagocyte recognition. Within herds, multiple strains can be isolated from clinical mastitis cases, although the isolation of dominant strains have a tendency to be responsible for chronic cases. 57 This suggests that the environment of the cow is an important reservoir of infection, as well as a limited number of host-adapted strains that can be transmitted both within-cow and between-cows, possibly at milking. 57 Early attempts by researchers in the United Kingdom to develop a vaccine for control of S uberis attempted the use of live vaccines in combination with an intramammary administration of a soluble cell extract. 58 A decrease in the development of clinical signs following experimental infection was realized, but the protection appeared to be strain specific. 58 Further research by the same research group determined that S uberis plasminogen activator, PauA, is an important virulence factor for acquiring nutrients in host tissue, and used this antigen for immunization in experimental infections. 59 Unfortunately, consistent clinical efficacy in large field trials and subsequent commercial application did not follow. More recently, reports have identified a S uberis adhesion molecule (SUAM), a virulence factor that enhances adhesion to mammary epithelial cells, and subsequent internalization. 60 SUAM appears to be highly conserved among strains from diverse geographical locations, and thus could serve as a potential universal antigen. 61 Vaccination of dairy cows with 3 doses of recombinant SUAM (rsuam), administered during the dry and peripartutient period, induced anti-rsuam antibodies in serum and colostrum at calving. 62 Furthermore, the antibodies were found to reduce adherence and internalization of S uberis into bovine mammary epithelial cells in vitro. 62 Although this technology offers promise, results demonstrating the impact of this vaccine on live challenge models and, more important, wide-scale field trials have yet to be published. Particularly because of the ability of S uberis to internalize in host cells, a successful vaccine against this pathogen will likely need to stimulate cytotoxic CD8 lymphocytes that can recognize and destroy bacteria-infected host cells, in addition to antibody responses. SUMMARY Presently, the most successful use of vaccination strategies as part of a dairy herd mastitis control program involves GNCABs, of which the J-5 bacterins are best understood. Immunization protocols employing this technology should be adapted to individual herd needs. Ironically, the success of these bacterins may rely, in part, on

11 Vaccination Strategies for Mastitis 267 the systemic pathogensis of severe coliform mastitis. Because immune function is impaired in the mammary gland of a lactating dairy cow, and the difficulty in maintaining effective concentrations of antibodies in milk following vaccination, vaccines developed against pathogens that cause more chronic IMI, while promising, have significant obstacles to overcome. REFERENCES 1. Rainard P, Riollet C. Innate immunity of the bovine mammary gland. Vet Res 2006; 37: Sordillo LM, Streicher KL. Mammary gland immunity and mastitis susceptibility. J Mamm Gland Biol Neoplasia 2002;7: Kehrli, ME, Harp JA. Immunity in the mammary gland. Vet Clin North Am Food Anim Pract 2001;17: Innate immunity. In: Murphy K, Travers P, Walport M, editors. Janeway s immunobiology. 7th edition. New York: Garland Science; p Netea MG, Van Der Graaf C, Van Der Meer JW, et al. Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system. J Leukoc Biol 2004;75: Park YH, Fox LK, Hamilton MJ, et al. Suppression of proliferative responses of BoCD4C T lymphocytes by activated BoCD8C T lymphocytes in the mammary gland of cows with Staphylococcus aureus mastitis. Vet Immunol Immunopathol 2003;36: Shafer-Weaver KA, Sordillo LM. Bovine CD8C suppressor lymphocytes alter immune responsiveness during the postpartum period. Vet Immunol Immunopathol 1997;56: Smith KL, Schanbacher FL. Lactoferrin as a factor of resistance to infection of the bovine mammary gland. J Am Vet Med Assoc 1977;170: Mullan NA, Carter EA, Nguyen KA. Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands. Res Vet Sci 1985;38: Preisler MT, Weber PS, Tempelman RJ, et al. Glucocorticoid receptor down-regulation in neutrophils of periparturient cows. Am J Vet Res 2000;61: Kehrli ME Jr, Nonnecke BJ, Roth JA. Alterations in bovine neutrophil function during the periparturient period. Am J Vet Res 1989;50: Waller KP. Mammary gland immunology around parturition. Influence of stress, nutrition and genetics. Adv Exp Med Biol 2000;480: Cai TQ, Weston PG, Lund LA, et al. Association between neutrophil functions and periparturient disorders in cows. Am J Vet Res 1994;55: Contreras GA, Sordillo LM. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comp Immunol Microbiol Infect Dis 2011;34: Rauff Y, Moore DA, Sischo WM. Evaluation of the results of a survey of dairy producers on dairy herd biosecurity and vaccination against bovine viral diarrhea. J Am Vet Med Assoc 1996;209: Cullor JS. The Escherichia coli J5 vaccine: investigating a new tool to combat coliform mastitis. Vet Med 1991;86: Tyler JW, Cullor JS, Spier SJ. Immunity targeting common core antigens of gramnegative bacteria. J Intern Med 1990;4: Tyler JW, Cullor JS, Dellinger JD. Cross reactive affinity purification of immunoglobulin recognizing common gram-negative bacterial core antigens. J Immunol Methods 1990;129:221 6.

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