Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  SAATK (VMRD, Inc., USA), designed to detect S. aureus intramammary infections by detecting antibodies in milk.

  Probably best placed as a screening test that is followed up with a confirmatory test. It has limitations on use based on stage of lactation and exudation of plasma in milk whatever the cause. See Section “Detection and Immune response to infection – Immunological basis of diagnosis”.

  GAP:

  Lack of a useful, rapid, specific cow-side screening test.

• Commercial diagnostic kits available in Europe

  Real-time PCR-based commercial reagent kit for detection of mastitis-causing pathogens: PathoProofTM Mastitis PCR Assay, ThermoFisher. This test may be ill-adapted to small ruminants.

  GAP:

  Rapid, specific, cow-side test not currently available.

• Diagnostic kits validated by International, European or National Standards

  No.

• Diagnostic method(s) described by International, European or National standards

  Standard bacteriological analysis of milk samples.

  Somatic Cell Count determination.GAP:Interpretation of species/strain identification by Mass Spectrometry (MALDI-TOF).

• Commercial potential for diagnostic kits in Europe

  Yes.

• DIVA tests required and/or available

  Not applicable at present.

• Opportunities for new developments

  The application of newer diagnostic methods such as PCR may allow the development of a veterinary lab based diagnosis as a first step in developing improved diagnostics.

• Vaccines availability

• Commercial vaccines availability (globally)

  None. Two vaccines based on killed whole bacteria (bacterins) are available: StartVac® (Hipra) in Europe and LysiginTM (HI) in US.

  GAPS:

  There is a need for effective vaccines.

  There needs to be a good definition of effectiveness for mastitis vaccines.

• Commercial vaccines authorised in Europe

  STARTVAC® (Hipra), based on killed bacteria.

• Marker vaccines available worldwide

  None.

• Marker vaccines authorised in Europe

  None.

• Effectiveness of vaccines / Main shortcomings of current vaccines

  Current vaccines primarily stimulate humoral immunity. Level of opsonising antibody in milk is poor or absent. These vaccines show some efficacy in decreasing the clinical severity of mastitis, but reduce the rate of new infection by 15-25% only. Review considering actual research data.

  GAPS:

  Lack of large field trial data.

  No therapeutic effect, which would useful to cure chronic infections.

• Commercial potential for vaccines in Europe

  There is a need for efficacious vaccines, because of the high prevalence of S. aureus mastitis in several European areas, and owing to the often poor cure rates obtained with antimicrobial treatments along with the necessary and required curb on the use of antibacterial agents in veterinary medicine.

  GAPS:

  The development of vaccines with good prevention of clinical mastitis and efficacy levels of >30% of prevention of chronic infections is needed.

  Definition of vaccine efficacy may vary based on regional differences in disease manifestation, i.e., subclinical versus clinical; chronic versus acute IMI; heifer versus cow.

• Regulatory and/or policy challenges to approval

  No.

• Commercial feasibility (e.g manufacturing)

  Feasible.

• Opportunity for barrier protection

  No.

• Opportunity for new developments

  Good if a more thorough understanding of the host-pathogen interaction and immunity to S. aureus can be elucidated.

  Vaccines are often seen as a means to reduce antibiotic use. The use of vaccines to potentiate antibiotic therapy by increasing the time the organism is limited to milk has not been fully evaluated in vaccine studies. As treatment efficacy can be as high as 80-90% in infections <2 weeks of duration, the use of a vaccine to extend this window to 4-8 weeks would greatly increase antibiotic efficacy and still reduce overall use.GAP:Investigate the possibility of therapeutic vaccine to cure chronic mastitis, either alone or in combination with antimicrobials. Previous unsuccessful attempts suggest that novel approaches are needed.

• Pharmaceutical availability

• Current therapy (curative and preventive)

  Numerous products marketed. Both lactating and dry cow therapies are widely available.

• Future therapy

  Improved therapies that utilize flexible treatments that are pathogen dependent will be needed in the future. The use of improved diagnostics and vaccines will be needed to maximize treatment efficacy. The use of peptide antimicrobials may offer the option of no withdrawal times, blanket fresh cow therapy and heifer treatments.

  Novel antimicrobial compounds that act intracellularly are under development.

  GAP:

  Most AH companies rely heavily on their parent firms to provide new antimicrobial agents. Since most companies have exited AB discovery, there are few compounds available to develop and lack of trained personnel in AH companies with experience in AB discovery.

• Commercial potential for pharmaceuticals in Europe

  Good, if proven efficacious.

  Low due to regulatory hurdles for new antimicrobial agents in veterinary medicine.

  GAP:

  Narrow spectrum antibacterials that are not considered critical for human use are needed.

• Regulatory and/or policy challenges to approval

  Current EU and US policies are hostile to the development of new antimicrobial agents in veterinary medicine.

• Commercial feasibility (e.g manufacturing)

  Feasible.

• Opportunities for new developments

  New classes of antimicrobial agents that provide high levels of efficacy with minimized zoonotic issues.

  GAP:

  Overall, the AH industry does not have active Antibacterial Discovery programs looking for new mastitis therapeutics.

• New developments for diagnostic tests

• Requirements for diagnostics development

  GAP:

  Rapid, sensitive, specific and cost effective laboratory, cow-side or in-line testing needed to help manage infected individuals in herds with S. aureus cattle.

• Time to develop new or improved diagnostics

  Unknown.

• Cost of developing new or improved diagnostics and their validation

  GAP:

  The biggest hurdle to development of a new diagnostic is cost. The cost per test must be very low.

• Research requirements for new or improved diagnostics

  See above.

• Technology to determine virus freedom in animals

  Not applicable.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  Entry level S. aureus vaccine should reduce the severity of clinical mastitis, reduce the incidence of new infections by >30%, and should increase the efficacy of antibiotic therapy.

  If possible, clear existing IMI (therapeutic vaccine).

  Cost-effective, easily stored and applied.

  GAPS:

  Improve the characterization of mastitis-causing S. aureus strains. For example, it could be useful to identify the major surface antigens and to examine the antigenic variation of proteins made by different clonal types. A whole-genome/proteomic approach to reverse vaccinology has resulted in some success for other organisms and would be a rational way to design an effective subunit vaccine by objectively testing the protective efficacy of surface-presented proteins.

  Other approaches such as characterizing the type of immune response most appropriate for protection may be useful. There are gaps in our knowledge of the identification, functions and regulation of lymphocytes in the ruminant species. This is now an active domain of research, but which needs to be reinforced.

  This knowledge could be put in practice by orienting the vaccinal immune response by using appropriate adjuvants.

• Time to develop new or improved vaccines

  7-10 years

  Vaccines against staphylococci have been studied for decades and while there are some promising candidates on the horizon, very few of the recently developed vaccines have made it to commercialization either in human or veterinary medicine. Hence, the likelihood of a new efficient vaccine reaching the marketplace in the near future is difficult to estimate.

  GAPS

  Owing to the lack of understanding of protective immunity and of the reasons why previous vaccines fell short of expectation, vaccine development resorts to hit-and-miss approach or to unproven rationales.

• Cost of developing new or improved vaccines and their validation

  R & D and eventual large scale testing would potentially cost millions (5-10) of Euros.

• Research requirements for new or improved vaccines

  New knowledge and tools are available to investigate the immune responses to S. aureus. This can give rise to the hope that some success will come despite previous failures.

  GAPS:

  There are a number of knowledge gap impeding the rational design of vaccinal strategy, involving both antigens inducing protection and vaccine-induced immune mechanisms protecting the mammary gland.

  Thorough understanding of the host-pathogen interaction is needed before proceeding with rational vaccine development:

  · Understanding of the diversity of S. aureus strains infecting dairy ruminants and the antigenic variation of surface-presented and secreted proteins;

  · Understanding of the molecular basis for pathogenesis of mastitis including genome-scale analysis of proteins involved in host-pathogen interactions;

  · Testing of selected antigens individually and in combination for ability to induce protective immune response;

  · Examine potential for conserved antigens as vaccine components;

  · Better knowledge of the (protective) immune response (cellular and humoral)- including host transcriptomic analysis of S. aureus infection;

  · Investment in basic research on cell-mediated immunity in the ruminant species;

  · New adjuvant favouring a protective long-lasting immune response.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  Depends on avenue taken. Identification of a new antimicrobial agent, if not available from Human Health program, may require development of veterinary specific analogous programme.

  GAP:

  Lack of personnel in AH companies with AB discovery experience.

• Time to develop new or improved pharmaceuticals

  Some novel antimicrobial compounds are currently under development, but require several years of in vitro and in vivo testing. May need 10-15 years.

  GAP:

  Lack of active AB discovery programs in AH companies.

• Cost of developing new or improved pharmaceuticals and their validation

  Potentially millions of Euros (30-50).

• Research requirements for new or improved pharmaceuticals

  Need to determine classes with necessary spectrum of action with reduced conflicts with uses in human medicine.

Disease details

• Description and characteristics

• Pathogen

  Staphylococcus aureus is a Gram positive pathogen that causes a variety of diseases in man and animals. In dairy cows, sheep and goats, the most economically important pathology caused by S. aureus is mastitis. This organism is a primary etiological agent of mastitis in man and animals.

  GAP:

  Although we know a great deal about S. aureus mastitis pathogenesis, all features of S. aureus which make this pathogen a successful parasite of the mammary gland have not been clearly identified. There is still a great deal to be learned about host-pathogen interaction of S. aureus in the context of bovine mastitis. The different genotypes of S. aureus have an important impact with regard to the proliferation of S. aureus (e.g. Genotype B is spreading easily within herds). So this gap does not exist any longer.

• Variability of the disease

  Considerable genetic and gene content diversity within the species. Existence of cow and sheep-specific clones of S. aureus suggesting a trend towards host-specialization. Different S. aureus strains infect humans, cows, sheep, poultry and pigs.

  In a given herd, mastitis isolates often belong to one or two dominant types or clones.

  GAPS:

  The epidemiology of S. aureus strains using modern genotyping techniques has not been applied on a large scale basis to determine the infection dynamics within and between herds.

  The different genotypes of S. aureus have an important impact with regard to the proliferation of S. aureus (e.g. Genotype B is spreading easily within herds). So this gap does not exist any longer.

  There are knowledge gaps in epidemiological characteristics of strains (contagiousness, clinical expression and flare up rates, curability, …). With regard to contagiousness and clinical expression research results have been published.

  Phase variation of S. aureus such as small colony variants (SCV) deserves more investigation in the context of mastitis.

  Importance of MRSA in bovine mastitis.

• Stability of the agent/pathogen in the environment

  The main reservoir in herds is constituted by infected mammary glands and skin/mucosae, but S. aureus can survive for some time in dairy cow environment (milking equipment and facilities). Strains of human origin (MRSA) are known to survive for weeks on fabrics and plastics. For the transmission of S. aureus Genotype B (often subclinical chronic) the milking equipment is the predominant way of propagation (milking order).

  GAP:

  The role of extra-mammary /environmental colonization as a reservoir for intramammary infection is not well-defined.

• Species involved

• Animal infected/carrier/disease

  Mastitis is the main disease caused by S. aureus in ruminants, including cows, sheep, goats, camels and water buffalo. Many other animal species can be affected by S. aureus, incl. horses, pigs, dogs, cats, rabbits and poultry. The pathogen may cause a wide range of conditions (see human infected/disease). Asymptomatic carriage is also observed in most species.

  GAP:

  Importance of between-host species transmission of S. aureus strains, including MRSA, has not been quantified. Cattle could be the source or recipient of MRSA in humans, pigs, and other species.

  Cattle have been shown to represent a reservoir for new human clones- requires further investigation to understand to what extent.

• Human infected/disease

  S. aureus is a major pathogen for human beings. This microorganism can cause a variety of pathologies such as skin and soft-tissue infections, sepsis, meningitis, pneumonia, endocarditis, septic arthritis, S. aureus is capable to produce a variety of toxins which results in severe food-poisoning: diarrhoea, vomiting, TSS (toxic shock syndrome). S. aureus is the most frequent pathogen isolated in cases of mastitis.

  GAPS:

  The role of mastitis-causing strains of animal origin as a reservoir for zoonotic infections seems limited at present but needs to be specified.

  The dynamics of transfer of S. aureus strains from humans to dairy ruminants and vice versa deserves to be investigated on a regular basis using modern genotyping methods such as whole genome sequencing.

• Vector cyclical/non-cyclical

  Flies have been shown to be colonized and act as possible vectors for the transmission of S. aureus in cases of bovine mastitis.

  GAP:

  The role of flies and other vectors as reservoirs of S. aureus within individual farms needs investigation using modern molecular epidemiological methods.

• Reservoir (animal, environment)

  The main source of S. aureus is infected mammary glands. S. aureus can colonize other body sites like the teat and inguinal skin, nares, and hocks particularly when wounded.

  GAPS:

  Specify the role of extra-mammary long-lasting contaminated locations (excluding fomites) as sources of contamination of the mammary gland, such as healthy persistent carriers at extra-mammary sites.

  Heifers are not exposed to the milking machine or the milking process, which is thought to be the main source of S. aureus. Environmental, insect, and extramammary infection sources and control measures in heifers are poorly understood.

  We also need to fully understand the transmission of S. aureus from humans to dairy animals (cows and small ruminants).

• Description of infection & disease in natural hosts

• Transmissibility

  S. aureus is a contagious mastitis pathogen that is primarily transmitted during the milking process. The bacteria are spread to uninfected quarters by teat cup liners, milkers' hands, wash cloths, and flies (fomites).

  See also Sears and McCarthy (2003).

  GAPS:

  Identify the main source of contamination of heifers prior to calving, in particular specify the role of skin colonization.

  Understanding of variable contagiousness among strains is lacking. Publications on the contagiousness of different genotypes are available.

• Pathogenic life cycle stages

  Not applicable.

• Signs/Morbidity

  In dairy cows, S. aureus mastitis is commonly subclinical, inducing raised concentrations of leucocytes in milk (elevated somatic cell counts). Most infections are chronic, persisting frequently over the ongoing lactation and possibly the following lactations, with more or less clinical flare-up episodes. Clinical cases show varying severity, from the infrequent gangrenous form to mild clinical (swelling, hardness, clots in milk). Severe forms (gangrenous signs) are much more frequent in goats and particularly sheep than in cows.

  GAPS:

  It is unknown to what extent manifestation (subclinical, mild, moderate, severe) and duration of infection are driven by host, pathogen, or management characteristics.

  What makes small ruminants more prone to severe S. aureus mastitis has not been identified.

• Incubation period

  Experimentally-induced infections even with low inoculum (< 100 cfu) exhibit short incubation periods (12-48 h) with most strains. Clinical signs usually occur early, but sometimes several days evolve before clinical manifestations. Incubation time is likely to depend both on the infected host and the infecting strain.

  Field monitoring suggests that incubation period until clinical signs is variable (days to weeks…). Infections may remain subclinical throughout their duration.

  GAPS:

  Based on experimental inoculation there seems to be tremendous cow-to-cow variation to a given infecting dose. In particular, early events taking place during the lag phase separating intrusion of staphylococci and the onset of inflammatory response are not well understood.

  Gaining insight into the temporal expression of the various virulence factors as infection progresses would be useful. This has a bearing on adaptation of S. aureus to the udder microenvironment and on in vivo expression of fitness and virulence genes.

• Mortality

  Direct mortality in bovine dairy herds is low. Indirect loss resulting in premature culling due to S. aureus incurable mastitis can be high in problem herds. Direct mortality can be high in heifers, ewes and goats due to peracute (gangrenous) mastitis.

• Shedding kinetic patterns

  Most infections are chronic with varying degrees of bacterial shedding (concentration of viable bacteria) in milk. Shedding is almost continuous with irregular patterns.

  Spontaneous cure with cessation of shedding does occur, although in a small percentage (< 20%) of cases of infections confirmed by several consecutive samplings. A lengthy dry period in ewes may favour spontaneous cure.

  GAPS:

  The impact of shedding patterns on mastitis diagnostics is poorly quantified.

  Diagnostic laboratories should include within their SOPs methods for detection and identification of those strains with atypical phenotype such as SCVs. Missing those colonies may be one of the reasons of the irregular pattern of shedding phenomenon.

• Mechanism of pathogenicity

  S. aureus mastitis is primarily a disease of ducts and alveoli, due to multiplication of staphylococci in milk and possibly on the mammary epithelium. Inflammation, which is triggered by non-specific response to staphylococcal MAMPs (Microbe Associated Molecular Patterns) and exoproteins (proteases and toxins), can be complicated by acquired immunity (hypersensitivity). Micro-abscesses may develop in the secretory tissue during chronic infections. The detrimental effects on mammary secretory tissue of an excessive inflammatory reaction are difficult to evaluate.

  The ability of S. aureus to invade and survive in mammary epithelial cells, demonstrated by in vitro experiments, is likely to have a major impact on chronic infection and resistance to antimicrobials.

  GAPS:

  All virulence factors have not been identified, and the mode of action of the putative virulence factors is not well understood.

  The contribution of the host responses to pathogenesis of mastitis is poorly understood.

  The possible role of SCV and biofilms in chronic subclinical mastitis needs investigation.

  The pathogenic potential of emerging so-called livestock-associated methicillin resistant S. aureus (LA-MRSA) strains has not been defined. Their contagiousness, pathogenicity and arsenal of virulence factors are unknown.

  The emergence of MRSA appears to be low in dairy cows relative to pigs but proper evaluation and surveillance is needed as it could be emerging problem.

• Zoonotic potential

• Reported incidence in humans

  Human infection caused by bovine-specialized clones is rare.

  GAP: Are immunocompromized individuals at risk?

• Risk of occurence in humans, populations at risk, specific risk factors

  Transmission to milking personnel by direct contact is probably frequent. Nevertheless, as most strains of ruminant origin are not well-equipped to induce disease in humans, the occurrence of disease in humans as a result of direct transmission from milk to humans is likely to be rare.

  Yet, a few isolates from bovine mastitis are related to methicillin-resistant S. aureus (MRSA) human strains, and exchange of genetic mobile elements between human and bovine strains is highly likely.

  A notable proportion of food poisoning cases is due to enterotoxigenic S. aureus contaminating milk or milk products. Part of these contaminations results from shedding of S. aureus by infected mammary glands. Foodborne infection risk is low as pasteurization is applied to most milk products, but there exists a risk with raw milk of insufficient quality and products made with raw milk (cheese). Also, enterotoxins are heat-resistant, hence toxin produced before heat-treatment can cause intoxication.

  GAPS:

  Need for continued epidemiological surveillance for emergence of strains common to both ruminants and humans. The flow of strains from humans to cows and the potential for cows/milk as a potential source of zoonotic infection needs additional investigation.

  The emerging LA-MRSA strains deserve a special attention.

  Global analysis of the capacity for transmission between different host species including humans using genome sequencing.

• Symptoms described in humans

  Food-poisoning through milk products- vomiting, diarrhoea TSS.

  Other pathology concerns mainly infections due to strains of S. aureus of human origin.

  GAP:

  Cutaneous and nasal colonization in humans with bovine strains is not well-defined nor whether exposure may lead to transient or persistent colonization or infection.

• Estimated level of under-reporting in humans

  Unknown, but food-poisoning is generally under-reported.

  GAP:

  Investigation of sources of food poisoning.

• Likelihood of spread in humans

  Spread of ruminant mastitis-causing strains among humans is low based on current knowledge.

  GAP:

  Needs surveillance of possible emerging strains such as LA-MRSA, as well as SCVs, supported by standardized methodology and recording and reporting infrastructure.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Impact of animal welfare depends on the form of the disease. The intensity of pain is difficult to assess, but is probably high in case of acute clinical mastitis. Chronic subclinical mastitis is likely to cause some discomfort.

  GAP:

  How mastitis affects the welfare of cows has not been properly investigated.

• Endangered wild species affected or not (estimation for Europe / worldwide)

  Not applicable.

• Slaughter necessity according to EU rules or other regions

  Not directly but S. aureus mastitis is a cause of premature culling, especially of small ruminants. Recourse to euthanasia remains infrequent but justified when gangrenous mastitis develops.

  In cases when herds are sanitized it is likely that cows that can not be treated , are culled.

• Geographical distribution and spread

• Current occurence/distribution

  Worldwide. Prevalence varying between countries (Intensity of dairying!).

  GAPS:

  Epidemiological surveys of bacteria responsible for clinical and subclinical mastitis are necessary worldwide to monitor the changing prevalence of S. aureus and the importance of this pathogen as agent of mastitis.

  Global analysis of the evolution and geographic spread of strains and the identification of new emerging strains using powerful genomic approaches.

• Epizootic/endemic- if epidemic frequency of outbreaks

  The herd is the epidemiological unit. Endemic in herds. Outbreaks can develop at the herd level, notably in early lactation in ovine flocks.

  Dairy cattle herds can be free of S. aureus intramammary infections for long periods (has to be reviewed taking in account research on contagiousness of different genotypes). Ovine and caprine flocks are seldom completely free of S. aureus infection, and clinical mastitis cases appear from time to time.

• Seasonality

  Climate does not seem to be a risk factor.

  When observed, seasonality is in fact related to other factors: calving periods, variation in risk factor exposure (flies for instance), variation of efficacy in prevention measures (seasonal implementation of teat dipping for instance), …

  GAP:

  Influence of heat stress on host susceptibility.

• Speed of spatial spread during an outbreak

  Spread between herds occurs mainly by introduction of an infected animal. Speed of spread within herds can be high, depending mainly on hygienic precautions implemented in the herd and during milking, as well as the virulence and transmissibility of the prevalent strains (genotype).

  GAPS:

  In some regions, there has been a move from closed herds to the use of contracted heifer farms that supply heifers to dairy farms. The movement of cattle between farms is much higher than 20-30 years ago and needs to be evaluated as a practice.

• Transboundary potential of the disease

  Possible but low incidence- directly related to the animal transfer!

• Route of Transmission

• Usual mode of transmission (introduction, means of spread)

  The portal of entry is the teat canal. Infected glands are the main source of pathogen. The microorganism is spread from quarter to quarter and from cow to cow usually during the milking process.

• Occasional mode of transmission

  Flies, skin, environmental reservoirs and fomites.

  GAP:

  Relative contribution of extramammary sources of infection needs to be quantified and control strategies developed.

• Conditions that favour spread

  Failure to follow basic hygienic milking procedures such as Schalm-test prior to milking (control of the milk quality of each quarter in regard of somatic cells) order of cows milked, use of adequately functioning milking machines, post-milking teat antisepsis, proper use of antibiotics and culling of refractory animals allow the organism to propagate in dairy herds. High prevalence (increases exposure), and poor control of risk factors (teat lesions and “over-milking” especially) favour spread within herds. Animal movements favour spread between herds.

  GAP:

  Cow, sheep or goat genetic susceptibility/resistance.

• Detection and Immune response to infection

• Mechanism of host response

  Once in the lumen of a healthy mammary gland following penetration through the teat canal, staphylococci multiply in milk. Bacterial compounds activate the innate immune system, which triggers inflammation. The initial immune response is characterized by an influx of neutrophils in mammary tissue and milk. Phagocytosis and killing of S. aureus by neutrophils is the main defence mechanism and is required to control S. aureus growth. Usually the initial neutrophilic inflammation is not able to clear the infection, which evolves in chronic mastitis. With time, foci of tissue fibrosis and/or abscess formation occur, and cell-mediated hypersensitivity may develop. This outcome appears to be host and strain dependent. Self cure occurs in a small proportion of cases.

  Infection of the mammary gland does not increase protection. Yet, antibodies are induced against several staphylococcal constituents. In particular, IgG opsonizing and toxin-neutralizing antibodies are induced, which increase the titres of antibodies pre-existing intramammary infection. Antibody and cell-mediated immune responses of the correct type and proportions are important in the context of chronic S. aureus mastitis. However, the precise adaptive immunological mechanisms that best control mastitis remain elusive.

  The genetic basis for resistance to mastitis is an active research field. In dairy sheep and goats, most infection are due to staphylococci. There are several QTL described for the susceptibility to Staphylococcus mastitis; few are common amongst breeds or species. Less is known in cattle where mastitis etiology is more diverse.

  GAPS:

  Opsonophagocytosis by neutrophilic polymorphonuclear cells has been shown to be an important arm of the immune defence against mastitis, but several areas need to be investigated. It seems worth:

  • Defining the role of cell-mediated immunity in protection and immuno-pathology.
  • Identifying the immune response(s) leading to clearing of mammary gland infection.
  • Specifying the role of toxin-neutralising antibodies in the control of infection.
  • Clarifying the role of antibodies neutralising staphylococcal adhesins.
  • Designing specific antigen/adjuvant/route of immunisation combinations to elicit the appropriate immune response.

  Some major genes, such as MHC, have been associated with mastitis risk, however their role in the context of other genes of lesser effect or genomic profiles is basically unknown. Therefore, more information is needed on the genetic basis for resistance or susceptibility to mastitis, with a view to identifying candidate markers or SNPs for selection.

• Immunological basis of diagnosis

  Anti-staphylococcal antibody titres increase as mastitis develops. Pre-existing antibodies are present in serum of all cows, against many S. aureus antigens. In milk of a healthy gland, antibody titres correlate with blood titres, due to transudation of plasmatic antibodies and to preferential transport of IgG1. In infected glands, milk titres depend more on exudation of plasma than on local synthesis. As a result, any inflammation of the mammary gland, such as streptococcal mastitis, provokes an increase in milk antibody titres to S. aureus, a phenomenon which complicates the use of antibodies for immunological diagnosis.

  Recruitment of leucocytes in milk by the innate immune system, a very sensitive marker of inflammation of the mammary gland, is a reliable means of detecting mastitis, although not specific to the causal agent.

  GAP:

  To find a S. aureus antigen inducing antibodies during infection, but not recognized by pre-infection serum (giving rise to sero-conversion). This goal may be unattainable.

• Main means of prevention, detection and control

• Sanitary measures

  Milking time hygiene (teat skin and equipment sanitation) has proven useful in decreasing the prevalence of S. aureus mastitis in dairy herds. However, in some situations these measures are inadequate at preventing spread. They have been recommended for the past 20 years and no new technologies have been introduced in that period.

  1. Prepare teats properly prior to milking. Udders should be dry, and teats should be cleaned and dried prior to machine attachment using single-service paper towels or individual cloth towels which have been laundered after each milking.

  2. Use adequately sized, properly functioning milking equipment. Use milking machines in a proper manner on properly prepared cows.

  Avoid unnecessary air admission into the teat cups during unit attachment, machine stripping and unit take-off that can cause irregular vacuum fluctuations.

  3. Disinfect teats. Use an effective product after every milking. Postmilking teat disinfection is the single most effective practice to reduce the rate of new intramammary infection by contagious pathogens.

  4. Assess clinical cases for treatment decisions. Most cases of clinical mastitis other than those caused by Streptococcus agalactiae, are only minimally affected by antibiotic therapy during lactation. Work together with the herd veterinarian to design a management protocol for mild, moderate, and severe cases of clinical mastitis. Treatment of subclinical cases may contribute to prevention of transmission. Response to treatment will be affected by host, pathogen and management factors, e.g. parity, duration of infection, penicillin resistance, and duration of treatment.

  5. Use dry cow therapy. Treat each quarter of every cow at drying off with a single dose of a commercially formulated, approved dry cow treatment product.

  For prudent use of antibiotics, selective dry cow treatment is a possibility that should be considered.

  6. Consider culling chronically infected cows. Cows which are infected with S. agalactiae, S. aureus, or Mycoplasma spp. present a risk to non-infected cows in the herd.

  7. Maintain a closed herd. If new animals are purchased, test them before adding them to the herd.

  8. Establish an active milk quality program with the herd veterinarian.

  9. Milk infected cows last, in a separate group, or with a separate cluster, or disinfect the cluster after milking of an infected cow.

• Mechanical and biological control

  Milk from infected cows should never come in contact with uninfected cows. S. aureus infected cows should be identified and milked last, or milked with a separate unit from those used on uninfected cows. Antimicrobial therapy during lactation or the dry period results in highly variable cure rates that are affected by host, pathogen and management factors. Clinical mastitis sometimes occurs following prolonged subclinical infections. S. aureus infection status of cows should be one of the considerations when culling decisions are made, as chronically infected cows are reservoirs of S. aureus. The most effective control is to prevent new infections by minimizing or eliminating conditions that contribute to the exposure of teat ends through spread of infections from cow to cow and conditions which allow bacteria to contaminate and penetrate the teat canal. In addition, certain nutritional components enhance the animal's resistance to mastitis. Supplementation of the diet with vitamin E and selenium, vitamin A and beta-carotene, and balancing dietary copper and zinc content to meet requirements have reduced mastitis.

  GAPS:

  Influence of nutrition on immunological status.

  Knowledge of genetics of resistance to S. aureus mastitis is scarce. Identification of markers of susceptibility, resistance or tolerance could be used in Marker-assisted selection (MAS).

  The impact on the infection outcome of the genetic and epigenetic modulation of the innate immune response deserves investigation.

• Diagnostic tools

  Diagnosis of inflammation is routinely performed by determining the concentration of cells (Somatic Cell Count) in milk samples. However, this is not specific for S. aureus intramammary infection.

  Etiological diagnosis is currently by bacteriological analysis of aseptically taken milk samples from individual mammary glands. The risk of contamination during sampling (cow-side conditions), phases of low shedding and time to get result are impediments to bacteriological diagnosis.

  Specific polymerase chain reaction (PCR) can be used to identify bacterial DNA in aseptically taken milk samples.

  Bacterial identification at the species (staphylococci) and even strain level can be greatly facilitated by using the mass spectrometry (MALDI-TOF), provided the data banks are updated for bacteria of animal origin.

  GAPS:

  Tests based on bacterial nucleic acids detection and quantification deserve research and development. Issues inherent to the technique (high sensitivity implying ‘sensitivity’ to sample contamination; inability to distinguish dead from live bacteria) will deserve due consideration.

  A rapid, cow-side or in-line, pathogen specific diagnostic kit would allow timely implementation of pathogen-oriented treatment. Diagnostics would need to be both sensitive and specific to avoid false-negative and, more importantly, false positive diagnoses.

  A monitoring system that will enable diagnosis on a frequent basis would ideally allow treatment of new infections within two weeks of occurrence.

• Vaccines

  Vaccines based on killed bacterins are currently approved in both the EU and US. Their efficacy is either limited and fickle or in need of large scale field evaluation. Numerous attempts to induce protection with subunit vaccines have been done or are ongoing, so far without convincing results.

  GAPS:

  There is a need for an effective S. aureus vaccine.

  Since the mammary gland is the predominant reservoir for contagious transmission, a vaccine that prevents intramammary infection or clears it very shortly after infection is needed. None of the vaccines studied to date have achieved this goal. Most vaccines rely on humoral immunity and humoral immunity in S. aureus infection prevention may not be sufficient since many hosts already possess a repertoire of anti-staphylococcal antibodies at the onset of infection.

  A careful evaluation of why previous vaccine attempts have largely failed would be useful. Recent progress in immunology, in particular cell-mediated immunity, should be taken into account to devise new approaches for development of effective vaccines.

• Therapeutics

  There are many antimicrobial agents approved for the treatment of mastitis in both lactating and dry cows. Antibiotic therapy during lactation may improve the clinical condition but usually does not eliminate infection. Dry cow therapy may give better results than treatment during lactation, but even then, chronic infections can persist into subsequent lactations. In vitro antimicrobial resistance of mastitis-causing strains is not the main reason of failure, and does not appear to be on the rise. Access of antimicrobials to infection foci (sequestration of alveoli by duct clogging, scar tissue barrier) and growth phase of staphylococci (intracellular position, small colony variants, microcolonies, biofilm) may contribute to this in vivo resistance. Chronic infections become more and more difficult to cure as time passes. Late diagnosis of infection, late onset of treatment, and short duration of therapy contribute to poor treatment outcomes. Standard commercial treatment is limited to 2-3 days whereas several scientific studies have shown that cure rates are higher with 5-8 day treatment.

  Dry-cow therapy is considered the most effective, but may also be unsuccessful, especially for long lasting infections.

  GAPS:

  Early detection of infection (including new subclinical cases) and etiological diagnosis would be useful, because current therapies are more efficient when applied in the first 2 weeks after inception.

  SCV may contribute to therapeutic failures.

  LA-MRSA strains are typically resistant to multiple antibiotics which are widely used in the treatment of mastitis. It would be useful to determine if these strains pose special treatment problems.

• Biosecurity measures effective as a preventive measure

  Milking time hygiene and monitoring of introduction of dairy animals in herds – See above.

  GAP:

  There is a paucity of data on the influence of purchased/imported cattle on herd biosecurity for S. aureus mastitis.

• Border/trade/movement control sufficient for control

  Not applicable.

• Prevention tools

  Milking time hygiene, teat disinfection, culling, segregation and separate milking, intramammary therapy. The main point is that control programs must reduce both the rate of new infections and the duration of existing infections.

  GAPS:

  Lack of an effective vaccine, antimicrobial efficacy and cost-effective rapid diagnostics somewhat hamper the application of prevention tools.

  Indicators of immunological status (background) could have a prognostic value and help for the decision to treat, wait or cull.

• Surveillance

  Individual cow or quarter or bulk milk somatic cell count monitoring and/or bacterial culturing.

  Surveillance of strains resistance to antimicrobials.

  GAP:

  Large-scale surveillance programs designed to determine the overall prevalence of mastitis pathogens including S. aureus not in place in most European countries.

• Past experiences on success (and failures) of prevention, control, eradication in regions outside Europe

  Eradication programs tend to be herd-based and in some herds have been successful. In other herds, maintaining a low prevalence of disease is the best that can be achieved.

  Country-level eradication does not seem to be attainable.

  Changes in regulatory limits on bulk milk SCC are reflected in changes in actual BMSCC. Uptake of existing control measures is improved when a regulatory incentive is in place.

• Costs of above measures

  10 to 25 €/cow-year;

  Somewhat herd and disease prevalence dependent, and also dependent on the approach used. Because many of the control measures detailed above are not exclusive to S. aureus mastitis control, actual costs can be difficult to discern on a pathogen basis.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  No.

• WOAH disease card available

  No.

• WOAH Terrestrial Animal Health Code

  Not available.

• WOAH Terrestrial Manual

  Not available.

• Socio-economic impact

• Zoonosis: impact on affected individuals and/or aggregated DALY figures

  Mastitis impacts on efficiency and profitability of milk production world wide.

  Mastitis limits the ability of farmers to invest time or funds in improvement of herd performance. Culling due to mastitis affects herd life of animals and opportunity for selection on other traits, e.g. productivity, persistence.

  GAP:

  Impact of S. aureus mastitis on carbon footprint of dairy production.

• Zoonosis: cost of treatment and control of the disease in humans

  GAP:No information available.

• Direct impact (a) on production

  Costs associated with mastitis include milk production losses, pharmaceuticals, discarded milk, veterinary services, labour, milk quality deficits, investment in mastitis management materials and infrastructure, diagnostic testing, and cattle replacement (Halasa et al., 2007).

  The following data are not specific to S. aureus mastitis. Estimates of the actual costs associated with cases of clinical and subclinical mastitis vary widely, are limited in the literature, and are somewhat herd and pathogen specific. According to a recent comprehensive review on the economic effects of bovine mastitis and mastitis management (Halasa et al., 2007), the cost per case of clinical mastitis was estimated at €287 and €102 per case of subclinical mastitis. In a UK study, the estimated annual output losses, treatment costs, and costs of prevention for mastitis were £197.9 million, £79.8 million, and £9.3 million, respectively (Bennett et al., 1999). These data underline the important economic impact of mastitis on the dairy industry, but when considering the costs of treating cases of mastitis, it must also be considered that treatment is not uniformly efficacious.

  GAP:

  Cost-benefit data to accompany efficacy trial data for vaccines, treatments, nutritional or management interventions.

• Indirect impact

  Possible “image” crisis related to multi-drug resistance strains in case of mention of cow-origin or presence in cows.

  Dairy products produced with milk containing S. aureus enterotoxins that need to be withdrawn from the market (including costs for dumping products and indemnity).

• Trade implications

• Impact on international trade/exports from the EU

  Varying milk somatic cell count limits worldwide potentially hamper international trade of milk and milk products from countries with higher legal limits to countries with lower legal limits. The legal cell limit in Europe is 400 000 cells/ml.

  Possible impact on the trade of cheese made with raw milk.

• Impact on EU intra-community trade

  No, except for raw- milk cheeses.

• Impact on national trade

  No.

• Main perceived obstacles for effective prevention and control

  - Sanitary and hygienic measures are labour-intensive, rapid, cow side, pathogen specific diagnosis is existing (PCR), and existing S. aureus vaccines have not a proven high efficacy.

  - Difficulty (no: somatic cell count) of early detection of mild clinical cases and, more frequently, new subclinical infections.

  - Limits of classical bacteriology (sensitivity defects, contaminated samples)

  - Segregation of infected animals not feasible in small herds.

  GAPS:

  Devise prevention practices adapted to robotic milking.

  Lack of incentive for uptake of control measures.

  Individual rapid diagnostic test may not be necessary and cost-effective for small ruminants.

• Main perceived facilitators for effective prevention and control

  The application of newer technologies to provide improved diagnostics as well as a better understanding of the immune response are needed to allow the development of improved therapies and vaccines.

  Importance of regular control of the milk quality by means of somatic cell count.

  In Switzerland experience exists in sanitation of herds from S. aureus Genotype B.

  GAPS:

  An effective vaccine.

  Economic, regulatory or social incentives for uptake of existing control measures.

• Links to climate

  Seasonal cycle linked to climate

  Climate does not seem to be a risk factor. When observed, seasonality is in fact related to other factors: calving periods, variation in risk factor exposure (flies for instance), variation of efficacy in prevention measures (seasonal implementation of teat dipping for instance)…

  GAP: Influence of heat stress on host susceptibility.

• Distribution of disease or vector linked to climate

  No.

  GAP: The role of flies in S. aureus transmission?

• Outbreaks linked to extreme weather

  Likely when teat chapping is induced by very low temperatures.

• Sensitivity of disease or vectors to the effects of global climate change (climate/environment/land use)

  GAP:

  Has not been identified.

Risk

• Given the complexities of the host-pathogen interaction and the general lack of efficacy of current treatment and vaccination strategies, there is always the risk of newly developed technologies failing to meet the needs of the dairy industry.

  Regulatory and political environment that is hostile to the development of new antimicrobial agents in veterinary medicine.

Main critical gaps

• • Improved understanding of the immunology of the mammary gland is needed. Current lack of understanding of how strain-to-strain variations (bacterial genetics and virulence factors) and host-pathogen interactions lead to different clinical outcomes. Recent progress in immunology should be taken into account to devise new approaches for development of effective vaccines.
  • Global analysis of the capacity for transmission between different host species including humans and global analysis of the evolution and geographic spread of strains and the identification of new emerging strains, including LA-MRSA.
  • Improved diagnostics: cheap, fast, specific, sensitive, cow-side/in-line
  • More information is needed on genetic basis for resistance or susceptibility to mastitis, with a view to identifying candidate markers or SNPs for selection.
  • Investing in antibacterial discovery programs to discover and develop new, more effective antibacterial agents for the treatment of S. aureus mastitis.

Conclusion

• Staphylococcal mastitis is a complex disease. S. aureus is a multifaceted pathogen which has the potential to express a myriad of virulence factors and is capable of evading immune surveillance and treatment compounds. These complexities are illustrated by the lack of efficacy of currently available vaccines and antimicrobial treatments. To effectively combat this disease a multifaceted approach must be taken. Control measures aimed at preventing S. aureus from entering the teat canal, namely milking time hygiene, has reduced the prevalence of this disease on many modern farms, yet the disease is still prevalent worldwide. In countries where dairying is a developing industry, it is likely that contagious mastitis caused by pathogens such as S. aureus and Strep. agalactiae may again become a prevalent disease due to lack of education or routine application of control measures. The fact that coagulase negative staphylococci (the most common bacteria isolated from milk) frequently carry antimicrobial resistance genes, such as mecA, that can potentially transmit to S. aureus is also concerning and surveillance of mastitis pathogens for antimicrobial resistance genes seems prudent.

  GAPS:

  Important gaps have been identified, which could be bridged by:

  • Searching the genetic arsenal of mastitis-causing strains to check whether the predominant clones share virulence factors which allow them to be successful parasites of the udder. To be complemented by studies on the expression of these genes in the infectious setting;
  • Investigating the strategies used by S. aureus to survive within the mammary gland and resist to treatments with antimicrobials: cell invasion, survival within phagocytes, biofilm or microcolony formation, SCV, …;
  • Investigating the basis for cow-to-cow variation in response to S. aureus mastitis: genetics of pathogen-specific resistance/susceptibility/tolerance, and influence of previous infection history (immune adaptive memory and innate imprinting);
  • Identifying protective immune responses, both those responsible for the observed spontaneous cures, and the vaccine-induced immune mechanisms;
  • Investing in Vaccine research and development: to identify protective antigens, to favour induction of protective immune responses (immunization schedule, adjuvant, …);
  • Investing in antibacterial discovery programs to discover and develop new, more effective antibacterial agents for the treatment of S. aureus mastitis;
  • Improved diagnostic methods (fast, cheap, sensitive, specific) to enable early detection and intervention through treatment or management;
  • Incentives for uptake of existing control measures.

Sources of information

• Expert group composition

  Expert group members are included where permission has been given

  Pascal Rainard, INRA Tours, France - [Leader]

  Gilles Foucras, Université de Toulouse, INRA, ENVT, Toulouse, France

  Gerrit Koop, Utrecht University, The Netherlands

  John R. Middleton, University of Missouri, USA

  Jeffrey Watts, Zoetis, USA

• Reviewed by

  Project Management Board

• Date of submission by expert group

  3 November 2016

• References