Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  Several commercial ELISAs and real-time PCR assays are available.Either as stand-alone tests targeting only M. bovis or “screening” tests targeting several agents potentially involved in Bovine Respiratory Disease (BRD) and mastitis including M. bovis (multipathogen kit).

  GAPS :

  Need for comparing the performance (sensitivity and specificity) and costs (dependency on reagents and laboratory consumables, time to perform) of the different kits through inter laboratory trials (only a few have been conducted so far).

  More investigations about methods to measure antibodies against M. bovis in bulk tank milk (BTM) are needed, on the model of what was done with the MilA ELISA recently (Salgadu et al 2022). Which commercial kits have been validated also for milk, with which performance? Similarly, kits for direct diagnostics of M. bovis from semen need to be validated.

  How can we use BTM antibody measurements in herd certification programs for instance? What is the ideal frequency for sampling to take into account intermittent excretion and the different stage of the production cycle, which impact on the tests results? Recommendations for “targeted” sampling are needed to maximize accuracy.

• Diagnostic kits validated by International, European or National Standards

  No.

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

  No. M. bovis is not included in the WOAH (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial animals.

• Commercial potential for diagnostic kits in Europe

  Yes. There is still room for improved, less expensive and faster kits.

  GAPS :

  Need for rapid (less than one hour) and cost-effective tests: Loop mediated isothermal amplification (LAMP), other isothermal PCRs or latex agglutination tests or lateral flow immunoassay devices.

  Pen side tests (both direct and indirect diagnostic) performable in the field and at local point-of-care (POC) centres would be also welcome. Real need to ensure exclusivity based on complex matrices. Field friendly DNA extraction (or direct to mastermix) methods (amplification amenable lysis solution that works at ambient) are needed for antigen field tests.

  Culture-based identification tests (like MALDI-TOF) should be encouraged as they allow strains isolation that are then available for antimicrobial susceptibility testing.

  Because M. bovis is a fast grower, accessibility to standardised growth medium in an easy to use format and controlled selectivity would be welcome.

  Methods for diagnostics should include antibiotic sensitivity testing and determining antibiotic resistance in real time. With many labs going down the direct PCR route to minimise costs this is increasingly constrained to specialist labs.

  Subtyping isolates might be pertinent in certain epidemiological situation (France for instance).

• DIVA tests required and/or available

  DIVA tests will be needed as soon as a new Protivity® vaccine is released (in France or worldwide), or for any other vaccine released.

  GAPS :

  Molecular DIVA test for direct detection could be based on genomic markers. It will be difficult to develop a DIVA test against the whole M. bovis bacterium for indirect detection (as very few antigens usually differentiate strains). It would be much easier to have an indirect DIVA test if the vaccine is composed of recombinant proteins.

• Vaccines availability

• Commercial vaccines availability (globally)

  Two bacterin-vaccines are commercially available in the USA (MpB Guard™ and Myco-B ONE DOSE™). Both showed moderate to low efficacy in reducing lung lesions but neither protected against M. bovis upper-respiratory tract colonization and otitis morbidity (Dudek et al .2021).

  In UK, there has been significant veterinary interest for importation of the US-manufactured 3 strains M. bovis bacterin vaccine: Myco-B (One Dose, American Animal Health, Grand Prairie, Texas,US). A first study showed a potential reduction in post-weaning mortality and use of antimicrobials ( https://issuu.com/senglobal1/docs/2021-iahj-winter-web_compressed_bae6584a52137c ).

  A new live attenuated (temperature sensitive, not a GMO) vaccine has been temporarily licensed for one year by Zoetis for use in France (Protivity®). Not yet available. Subcutaneous inoculation route has been chosen for ease of use. Main target = BRD with some promising results during an experimental challenge.

  GAPS :

  So far “off-label” vaccination program, limited recent trials.Efficacy and safety of Myco-B has still to be demonstrated.

  Protivity® efficacy in the field has yet to be demonstrated. More data are needed about the origin of the strain. The vaccine strain was developed from an Australian wild type M. bovis strain. It was originally tested in aerosol vaccination, but later Zoetis has tested it as a subcutaneous vaccine. The challenge strain used in their studies was an US wild type strain.

  The subcutaneous route using an attenuated strain might be questioned in terms of immune response and efficient replication of the vaccine strains once in the animal.

• Marker vaccines available worldwide

  See Section above “Commercial vaccines availability”.

  Autogenous vaccines are used in different countries.. Most often they give the impression of positive results, but no real challenges have been conducted. Effects seem to be limited without additional improvements in herd management / housing.

  GAPS :

  Autogenous vaccines: Measuring antibody level post vaccination does not give an insight into their protective effect and data about clinical improvements are most often missing in experiments.Field trails would be lengthy and expensive (who is willing to fund?) but research on autogenous vaccine might not be discouraged.

• Effectiveness of vaccines / Main shortcomings of current vaccines

  Most of previous vaccine studies have been based on single strain bacterin preparations (Maunsell et al. 2009). Using a multiple strain vaccine like Myco-B, has previously been suggested as a way of improving M. bovis vaccine efficacy (Calcutt et al. 2018). But strains should be selected to best encompass diversity (and ideally global diversity).

  GAPS :

  It has been suggested that vaccine development should be based on conserved recombinant proteins, not bacterin-based vaccines (Perez-Casal at al. 2017); trials using recombinant protein vaccines have been unsuccessful to date (Prysliak et al. 2018).It is important to take into account the targeted animal population in vaccine developments: beef versus dairy cattle, age of the animals. For instance, bacterin-vaccine showed some efficacy on bison (informal discussion with David Hunter, former head veterinarian of Ted Turner’s bison operation in the USA).

• Commercial potential for vaccines in Europe

  Combined vaccine including other etiological agents of BRD will be welcomed by breeders.Trouble with mycoplasmas’ vaccines: the presence of other, potentially close, commensal species.

  Several experimental vaccines have been developed as summarized in a recent review by Dudek et al. 2021, but none has been brought to a commercial format.

  GAPS :

  How far can we go in combining strains or proteins in a vaccine preparation? What is realistic versus really needed?

  Proteomic analysis of M. bovis secretome (Zubair et al. 2020) has brought forward some potential candidate proteins for future vaccine development.

  There are 4 steps in vaccine development: 1/ understanding of immune response, 2/ choice of the antigens, 3/ choice of the adjuvant and 4/ choice of the route of immunisation. Still a long way to go for M. bovis.

  Needs for developing an animal challenge model. For BRD, an aerosol challenge model has been proposed in Australia (Kanci et al. 2017).

  Animal challenge models for feedlot cattle (6 to 8 months-old) were developed (Prysliak et al. 2011; Perez-Casal, unpublished). Inna Lysnyansky's and Nahum Shpigel's groups developed a mouse mastitis model, but not clear how it would compare to a cow model. Use of sheep for mastitis model development may be cheaper.

• Regulatory and/or policy challenges to approval

  The country of origin of the strain used to produce the Protivity® vaccine (attenuated through mutations) might be a barrier for worldwide market.

  GAPS :

  Because the Protivity® vaccine is based on an attenuated vaccine strain, with a risk of dissemination, we need the development of DIVA tests to control potential spill-over or reversion.

• Commercial feasibility (e.g manufacturing)

  Intranasal route might be more efficient than subcutaneous route but not easily doable, hence less easily saleable. However, intranasal administration is also a common practice in feedlots.

  GAPS :

  Needs for research about the best route of administration. Intranasal route is also worth trying in dairy cattle where animals are gathered daily for milking.

  Need to develop other commercial adjuvants.

  M. bovis vaccine should be part of a wider vaccination program involving other respiratory pathogens, including for instance BVD, PI3V, Mannheimia, Pasteurella and possibly others.

• Opportunity for barrier protection

  Biosecurity, restrictions in importing animals. For importation restriction, we probably need some standardization (should be animals free by ELISA, PCR or both).

  GAPS :

  A herd and/or zone certification programme could be useful in some areas but how to show freedom or at least low risk reliably? (Risk of introduction through germplasm?)

• Pharmaceutical availability

• Current therapy (curative and preventive)

  There is a poor response to treatments against M. bovis in both respiratory disease and mastitis. A range of antibiotics (ATB) have marketing authorizations worldwide and are currently used against M. bovis (except for some molecules of critical importance, like fluoroquinolones). However, decreased susceptibilities in vitro have been described worldwide for many families (Pereyre & Tardy 2021).

• Future therapy

  Need for new or cocktails of antimicrobials that are effective against mycoplasma and used rapidly once the infection is in a herd. Presumably for mastitis form these antimicrobials would also need to be rapidly cleared from udder tissues.

• Commercial potential for pharmaceuticals in Europe

  GAPS :

  ATB sales are receding in Europe and the USA but increasing in other parts of the world: a market exists. However, currently development of new antimicrobials is not seen as socially responsible (what is the point of bringing to the market a new molecule that will have resistance one year later?) and hence not a priority for many industries.

  Probiotics and medicinal plants effectiveness should also be considered.

• Regulatory and/or policy challenges to approval

  None.The pressure to reduce antimicrobial use may be a policy challenge.

• Commercial feasibility (e.g manufacturing)

  Feasible, but needs to find its market: veterinarians must think of M. bovis as an etiological agent of BRD and not wait until failure of first line treatment to use molecules with also an anti-mycoplasma efficacy.

• New developments for diagnostic tests

• Requirements for diagnostics development

  Diagnostics should be standardized, fast and cheap.Antimicrobial resistance testing should be included in the future tests. DIVA tests are also needed as soon as a new vaccine is marketed.

  GAPS :

  Comparison and critical evaluation of existing diagnostic tests are required worldwide.Definition of the most suitable type of samples and time of sampling is also important.

• Time to develop new or improved diagnostics

  New diagnostics should be rapid (lots of samples available and other tests usable as reference), i.e. less than one hour. On-field diagnostics would be welcomed as well.

• Cost of developing new or improved diagnostics and their validation

  Depends on the nature of the test.

• Research requirements for new or improved diagnostics

  Increased knowledge on virulence and pathogenesis.Increased knowledge on cell invasion process.Increased knowledge on the host immune response and immunity during chronic forms.

• Technology to determine virus freedom in animals

  Multiple samples (due to intermittent shedding) and repeated tests with a very low limit of detection.

  GAPS :

  Beware of intracellular localization of M. bovis. The choice of the sample and the sample treatment before culture or DNA extraction need to be considered in estimating the performance of a diagnostics.Beware of bacteriostatic effect of antibiotics in extended bovine semen – several dilutions needed if culture is attempted.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  Increased knowledge on the host immune response and immunity during chronic forms.Studies about the best adjuvants and the best inoculation routes.Need for good (cheap and easily feasible) challenge models.

  GAPS :

  Vaccines are needed as chemotherapy is rather ineffective and may lead to carriage, with potential for intermittent shedding.

• Time to develop new or improved vaccines

  Two EU-funded projects (Mycosynvac and Saphir) failed to deliver new ready to use M. bovis vaccines (combined with Bovine Respiratory Syncytial Virus for Saphir) but some advances in scientific knowledge (mechanisms of immune protection and pathogen invasion; impact of age and host genetics, optimization of adjuvant and formulation for rapid, widespread and long-term immunity; induction of mucosal immunity).

  GAPS :

  Need for high TRL projects call.The “chassis” based vaccine (as proposed by the Mycosynvac project) is promising: expressing some antigenic proteins of M. bovis in a fast growing chassis with no virulence left.The importance of exopolysaccharides or glycosylated proteins for leveraging a good immunity needs to be considered.The production of extracellular vesicles by mycoplasmas might be also of interest for vaccine development (choice of antigens) or at least as an adjuvant (Gaurivaud & Tardy 2022).

• Cost of developing new or improved vaccines and their validation

  Need for high Technology Readiness Level (TRL) project calls.Need for multi-target vaccines (either target a syndrome and its different etiological agents) or using the same targeted proteins in different Mycoplasma species (e.g. pig and ruminant industries).

  GAPS :

  There is still a long way to go in understanding the immune response against mycoplasmas and hence be able to develop the appropriate (efficacy and safety) vaccines.

• Research requirements for new or improved vaccines

  A lot with low TRL so far. Most industries are expecting academic partners to bring in projects where the efficacy of the vaccine in the target species has already been demonstrated.

  GAPS :

  Research on autogenous vaccine that might open some opportunities must be encouraged.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  GAPS :

  Screening of novel chemicals and plant extracts is required to develop new pharmaceuticals. Proper regard will need to be given to meat and milk withdrawal times for newly developed pharmaceuticals. Major mechanisms are known. Others need more works, i.e. efflux.Interpretation criteria based on pharmacodynamics and kinetics are required to interpret inhibition in vitro in terms of chance of therapeutic success.

• Time to develop new or improved pharmaceuticals

  Time to develop would depend on the product and the trials necessary to validate the efficacy and safety (already 12 to 15 months with regulatory matters with the EMA in Europe). Commercial production would then take supplementary time. Most industries are expecting profits within 5 to 10 years post development of a new molecule.

  GAPS :

  Efficacy of cocktails of antibiotics (synergetic effects) to be studied.

• Cost of developing new or improved pharmaceuticals and their validation

  Expensive but difficult to assess as it will depend on the product and the trials necessary to validate and license.

• Research requirements for new or improved pharmaceuticals

  Standardization of MIC tests.Determination of MIC breakpoints for Mycoplasma bovis.Decoding mechanisms responsible for antibiotic resistance.Identification of relevant protective antigens, through genomic, bioinformatic, proteomic, immunological and biological approaches.

  GAPS :

  Rapid and improved minimum inhibition concentration and mycoplasmacidal tests need developing and standardising. Clinical breakpoint values need to be determined for most antimicrobials against M. bovis to relate in vitro tests to in vivo situations. These tests should take into account the possible impact of biofilm formation. The development of resistance and mechanisms of resistance will also need investigating.

  Choice of adjuvants, routes of administration have to be explored further.

Disease details

• Description and characteristics

• Pathogen

  Mycoplasma bovis is a member of the genus Mycoplasma and family Mycoplasmataceae within the Class Mollicutes. Mycoplasma bovis was first detected as a cause of bovine mastitis in the USA in the 1960s and has since been detected in most countries worldwide. New Zealand that used to be one of the few free countries was infected in 2017.

  GAPS :

  Host-pathogen interaction.Modulation of the immune system.

• Variability of the disease

  Mycoplasma bovis is considered to be one of the most pathogenic species of Mycoplasma and is an important pathogen of cattle. It is responsible for pneumonia, arthritis, otitis and mastitis.

  Genomic data has largely increased recently and are helpful to monitor epidemiological spread BUT not to understand the physiopathology or decipher virulence factors of M. bovis.

  PubMLST scheme allows to have a nice overview of the current subtypes diversity worldwide (https://pubmlst.org/organisms/mycoplasma-bovis). Another relevant subtyping scheme relies on the polC sequence gene (Becker et al 2015).

  GAPS :

  Mycoplasma bovis disease makes a significant economic impact on cattle rearing, but its importance has not yet been recognised sufficiently to be listed by The World Organisation for Animal Health (WOAH).Need for understanding the organism’s mechanisms for survival and its evasion of the host defence mechanisms. Would genome-wide association studies bring an answer? Not sure due to poor annotation and poor functional genomic data.Making mutant libraries to understand the essential genes for its survival in media and investigating which genes are essential for survival and invasion of the host cells through in vitro cell models, coupled with omics approaches to understand genes metabolic functions.

• Stability of the agent/pathogen in the environment

  Mycoplasmas lack a cell wall that should make them susceptible to environmental pressures, however one report has suggested they could survive for long periods (bedding sand for 8 months) (Justice-Allen et al., 2010).

  Existence of non-bovine reservoirs has been suggested by re-emergence of receding genotypes (Tardy et al 2020).

  GAPS :

  The survival in biofilms or in farm environmental samples has yet to be further demonstrated. Potential for transmission to naïve hosts from environmental samples have not been demonstrated.

• Species involved

• Animal infected/carrier/disease

  Infected cattle can become asymptomatic carriers and may shed the organism through nasal discharges or in milk for months to years without showing clinical signs. It is the most frequent Mycoplasma pathogen linked to pneumonia, mastitis, and arthritis in cattle. Semen has been recently demonstrated to be a source of contamination (Haapala et al. 2018).Affect all production sectors (although not equally in all affected countries).Bison and buffaloes can also be affected.

  GAPS :

  Actually, there is no "common flow" what to do to detect asymptomatic carriers, especially nasal carriage.

• Human infected/disease

  Not applicable.

• Vector cyclical/non-cyclical

  Not applicable.

• Reservoir (animal, environment)

  Cattle, bison, buffalo, mule deer, pronghorns.

  GAPS :

  Certainly, other potential reservoirs. Regularly isolated from sheep and goats but often in farm setting where sheep/goat and cattle are bred in close proximity.

• Description of infection & disease in natural hosts

• Transmissibility

  Highly contagious in high stocking density. Easily transmitted to close contacts often by aerosol. Mastitis is mainly udder-to-udder spread through indirect contacts.

  GAPS :

  Other routes of transmission need to be investigated further (milk, colostrum, semen, intrauterine route, milking equipment, etc…) as well as the infectious dose.Do we need zero tolerance in artificial insemination semen?

• Pathogenic life cycle stages

  Not applicable.

• Signs/Morbidity

  Variable with 3 dominant ones: mastitis, pneumonia, arthritis. In Europe pneumonia of young animals is the main M. bovis associated sign but mastitis can be also a problem elsewhere (USA, Israel). Otitis media (young calves) and brain lesions are seldom described and can be considered secondary to pneumonia.Mastitis can be chronic, subclinical or clinical, up to severe forms with one or all quarters being affected with a serous or purulent exudate.Calf pneumonia with M. bovis is typically a non-specific respiratory disease that does not respond to antibiotics and is nearly always associated with a range of other pathogens where it may have a synergistic role. Similarly, arthritis is associated with an acute inflammatory response in joints and can be difficult to treat successfully with antibiotics.Different types of signs are often co-existing in an animal: for example, mastitis with arthritis or pneumonia; in calves - pneumonia with arthritis or otitis media.

  GAPS :

  Beware of not overlooking M. bovis in pneumonia cases because of the presence of more familiar pathogens. Veterinarians have to take M. bovis into account as a potential etiological agent in antibiotic choices!

  Similarly, M. bovis in mastitis might be not searched for in some countries.

• Incubation period

  Variable depending on the age of animals, the presence of other co infecting agents, the herd type and management.Experimentally, incubation may be a few days for mastitis and from 7 days on for pneumonia.Field longitudinal monitoring studies are less accurate about the time of introduction of M. bovis in the herd and less controlled about concurrent risk factors but nonetheless spread is very rapid. A recent paper (Catania et al. 2020) showed that the prevalence of M. bovis in fattening farms increased from 1-2% at the day of introduction to 81% after 15 days as detected by PCR. In one herd with an outbreak of mycoplasma mastitis the incubation period was estimated to be 13.6 days (Punyapornwithaya et al., 2011).

  GAPS :

  The strain and herd management differences may impact on the length of the incubation period making this a relatively unknown factor in the control of the disease.Of note, estimating the incubation period in wild animals is even more difficult.

• Mortality

  Variable depending on the clinical disease, age of animals and other infections. Can be high in the case of pneumonia and arthritis. Mastitis often leads to culling. Arthritis is associated with a high morbidity.

• Shedding kinetic patterns

  Infected cattle can shed the organism for months to years.Shedding in milk is intermittent (Reviewed in Maunsell et al. 2011).

  GAPS :

  Role of the asymptomatic carrier in a herd outbreak is largely unknown.

• Mechanism of pathogenicity

  M. bovis can probably invade tissues and enter the bloodstream to spread to other tissues.Mechanisms of pathogenicity are currently poorly understood for M. bovis. Its transmission within the host, predilection for specific sites, intermittent shedding and differences in resulting clinical signs are not known. The role of some defined virulence factors such as the variable surface proteins in disease is still to be ascertained. Possible differences in route of infection, infectious dose, host susceptibility, age, breed etc. also require investigation. The ability of M. bovis to invade non-phagocytic cells such as lymphocytes, epithelial and synovial cells (van der Merwe et al. 2010; Bürki et al. 2015; Suleman et al. 2016; Nishi et al. 2021) needs further study. The invasion of circulating immune cells and erythrocytes could play an important role in pathogenicity of Mycoplasma bovis disease (protection of the pathogen from host immune response, administered antibiotics and may lead to persistence of infection and dissemination of the pathogen between organ systems).

  GAPS :

  Lack of experimental model for reproducing the disease. A mouse model has been proposed for mastitis (Schneider et al., in preparation).

  Complex cellular systems are more and more used to study the interaction of M. bovis with the host cell. Effectiveness of tissue explants e.g. Trachael explants – recently least for CBPP; most work on M. bovis is from the 80’s (Di Teodoro et al. 2018). No publication yet on miniorgans.

  Role of the recently described releasome (including extracellular vesicles) in virulence to be further studied (Recent review by Gaurivaud & Tardy 2022).

  Need for clarification about the way M. bovis invades its host cells. This is essential for understanding the pathophysiology and could hint towards new therapeutic development. Recently, a clathrin-dependant endocytosis pathway was suggested for synovial cells invasion (Nishi et al. 2021) but other pathways could co-exist.

  Exploring the interactions between the host immune system and M. bovis especially during mastitis and arthritis.

• Zoonotic potential

• Reported incidence in humans

  No.

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

  No.

• Symptoms described in humans

  No.

• Likelihood of spread in humans

  Very weak.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Arthritis, acute or chronic, has an important impact on animal welfare. Severe inflammatory reactions in joint might lead to animals culling to avoid suffering.No wild animals are slaughtered (ethical debate). Pasteurisation or heat treatment to eliminate the risk of M. bovis shedding via colostrum or raw milk, as well as avoidance of colostrum from affected cows are good practices, but may have adverse effect on cytokine absorption and immune response when fed to neonatal calves.

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

  Buffaloes from national parks are endangered. So are pronghorns and mule deer.

• Slaughter necessity according to EU rules or other regions

  No.

• Geographical distribution and spread

• Current occurence/distribution

  Distributed worldwide with a few exceptions of countries with unknown status (e.g. Norway). It is the most important Mycoplasma pathogen in cattle in the USA and Europe.

• Epizootic/endemic- if epidemic frequency of outbreaks

  Can spread very rapidly once introduced into a herd, especially naïve or a country (see Northern Ireland (Reilly et al. 1993) and New Zealand ( https://www.mpi.govt.nz/biosecurity/mycoplasma-bovis/what-is-mpi-doing/history-and-background-to-the-eradication-programme/#how ).The introduction of the disease in a herd is due to the movement of asymptomatic carriers being purchased and introduced into a clear herd or through semen (Punyapornwithaya et al. 2010; Haapala et al. 2018).

  GAPS :

  Suspect that stress of cattle (climatic changes, overcrowding, introduction of new animals, and translocation) might trigger an outbreak.

• Speed of spatial spread during an outbreak

  Rapid (with or without seroconversion of all animals in a herd).

• Transboundary potential of the disease

  High, association with movement of clinically normal infected animals in the absence of any control + germplasm (semen, ova and embryos) export.

• Route of Transmission

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

  The primary routes of infection can vary depending on the problem in the infected herd but are usually close contact through direct nose-to-nose transmission via aerosols and/or by the ingestion of infected milk, or shared feeding systems +milking parlour hygiene for mastitis + semen (recently demonstrated).

  GAPS :

  Intracellular role, bacteraemia and spread of Mycoplasma bovis which may be associated with the different clinical signs observed in animals, including arthritis, pneumonia, mastitis, and keratoconjunctivitis.

  Survival in the environment is poorly documented as a route of re-infection.

• Occasional mode of transmission

  Ingestion of contaminated milk is a major source for calves.

• Conditions that favour spread

  Density of animals, size of the herd, feeding systems (Arcangioli et al. 2021).Movement of infected animals into clean herds or vice-versa.Increased risk of mastitis linked to larger herds.

• Detection and Immune response to infection

• Mechanism of host response

  Humoral response. Immune response contributes to the lesion development, at least at chronic stage.

• Immunological basis of diagnosis

  Serological tests for the presence of antibodies. Immune response seems to last much longer than the clinical disease/molecular detection; the problem is we don’t know if the immune response detection is due the response lasting longer or due to animal harbouring the bacteria and becoming a carrier. Not yet clear how we can separately diagnose them.

• Main means of prevention, detection and control

• Sanitary measures

  Mycoplasmas can be introduced in a herd by subclinical infected carriers. Once established in the herd, the infection is difficult to control.No possibility to control carriage.

• Mechanical and biological control

  Limited methods available for control. Control of other pathogens by vaccination to reduce the impact of M. bovis as a secondary infection. When Mycoplasma bovis is the primary pathogen it can be difficult to control. Preventing the introduction into the herd by sourcing replacement stock from known free herds, management factors by avoiding mixing cattle of different ages especially calves and culling positive animals. Reducing co-mingling stress can reduce clinical presentations, both for mastitis or respiratory disease.

• Diagnostic tools

  Isolation and identification of M. bovis from bulk milk tank or from cows with clinical mastitis, respiratory specimens. Use of M. bovis specific PCRs, real time PCRs and microarrays is increasing. Use of the 16S rDNA PCR and DGGE (denaturing gradient gel electrophoresis) detects and differentiates the many Mycoplasma species detected in cattle.Serology using paired sera collected at 10-14 day intervals to detect rising antibody titres. Many different tests have been used including indirect ELISA, indirect haemagglutination etc.

  GAPS :

  Intermittent shedding of organisms and the inhibitors present in milk may reduce the efficacy of current tests.Isothermal amplification tests such as LAMP are being reported, but are not in routine use, particularly in the field where sufficiently simple DNA extraction can be very challenging.Some workers describe the use of MALDI-TOF, also with only a rapid enrichment procedure (Bokma et al. 2020). No pen-side tests are currently available.

• Vaccines

  A number of commercial vaccines exist prepared from a limited number of strains.

  GAPS :

  Autogenous vaccines are produced by several companies in different countries. Data about their efficacy is sparse. Their conditions for productions have not been harmonized yet in Europe. Research needs to be addressed at the development of live vaccines.

• Therapeutics

  Mycoplasma bovis as with other mycoplasmas lacks a cell wall, which means the organism is resistant to some commonly used antibiotics. In general Mycoplasma bovis is resistant to antibiotic therapy which can also be expensive and ineffective.France has reported that an antibiotic resistant molecular type is now the dominant strain present in France.

  GAPS :

  Several countries have reported antibiotic resistance by Mycoplasma bovis to many antibiotics, including macrolides, tetracyclines, lincosamides, aminocyclitols and fluoroquinolones. Some mechanisms of resistance have been determined as similar to other bacterial species, however some mechanisms have not yet been discovered and require further investigation. Efflux mechanisms are one possible area for investigation.

• Biosecurity measures effective as a preventive measure

  Limited effect, but a closed herd policy preventing the introduction of Mycoplasma bovis into a herd is important along with general measures to reduce the levels of infection in the environment. Avoid mixing calves of different age groups. Some recommend distancing dairy farms from calf fattening units. New Zealand has produced several recommendations on biosecurity measure to control M. bovis in their attempt to eradicate the disease https://www.mpi.govt.nz/biosecurity/mycoplasma-bovis/what-is-mpi-doing/

• Border/trade/movement control sufficient for control

  New Zealand has managed to control their outbreak effectively so far. They re-evaluated and re-established all their border/trade movement policies as part of the eradication program.

• Prevention tools

  Extensive herd management.

• Surveillance

  Some surveillance systems do exist although they are passive (France and UK, for instance, Deeney et al. 2021; Jay et al. 2021).For eradication purpose, New Zealand has developed a complete monitoring system of the disease (https://www.mpi.govt.nz/biosecurity/mycoplasma-bovis/what-is-mpi-doing/ ).

  Others:Serosurveillance and disease surveys can be undertaken.

  GAPS :

  The prevalence seems to vary considerably by country. In the US and Canada the prevalence may vary from 2% to >30%, depending on the region. Mexico’s herd prevalence appears to be higher. The true incidence of Mycoplasma bovis is not known, available information appears to be based on passive surveillance information. A serological survey and abattoir survey could give more information about its true prevalence. The real economic cost of the disease has not been determined – a survey to include all cost factors that includes mortality, veterinary costs, treatment, milk loss, added housing/feed costs through lack of weight gain etc would provide useful evidence.

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

  Eradication of M. bovis from New-Zealand after a late introduction (2017).

• Costs of above measures

  Very expensive. Culling can be devastating and the use of antibiotics is expensive.

  GAPS :

  Reports indicate that early recognition of the disease and prolonged therapy is required and consideration should be given to metaphylaxic treatment of whole groups.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  No.

• WOAH disease card available

  Not available.

• 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

  Non-zoonotic.

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

  Non-zoonotic.

• Direct impact (a) on production

  Important. M. bovis is responsible for an “economic” disease.

  GAPS :

  Lack of recent cost assessment.

• Direct impact (b) cost of private and public control measures

  Low (no regulatory constraints).

• Indirect impact

  Medium.

• Trade implications

• Impact on international trade/exports from the EU

  No regulation except for some countries (New Zealand).

  GAPS :

  Harmonisation of control of M. bovis presence in semen would be a plus worldwide. Bovine Semen for export to New Zealand is now required to be screened using stipulated PCR tests (Bovine Germplasm Import Health Standard (mpi.govt.nz). More information is needed as to prevalence in semen of microbiologically or seropositive animals and whether the organism has the capability to invade sperm cells (just attaching to acrosome or some other part or going inside sperm cells. There is one study indicating that Ureaplasma diversum can invade sperm cells).

• Impact on EU intra-community trade

  No EU regulation so far.

• Impact on national trade

  No regulation.

• Main perceived obstacles for effective prevention and control

  • No effective vaccines available,
  • Insidious infection not always easily diagnosed
  • Difficult to eliminate from a herd
  • Difficult to assess cause of the bovine respiratory disease complex when a number of other pathogens are also involved.
  • Development of antibiotic resistance to many of the antibiotics currently in use.

• Main perceived facilitators for effective prevention and control

  Effective vaccines.

  GAPS:

  • Effective vaccines.
  • Reproducible vaccine challenge methods.

• Links to climate

  Seasonal cycle linked to climate

  For countries where seasons are not drastically different, it follows the milk production cycle (Salgadu et al. 2022). In Finland where the 4 seasons are still marked, respiratory diseases are at peak from January to March but no seasonality is observed for mastitis.

• Distribution of disease or vector linked to climate

  No.

• Outbreaks linked to extreme weather

  No, apart from the impact of adverse weather on associated stress on the animals.

  GAPS :

  Would be interesting to follow up: seasonality is influenced by climate change.

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

  Intensive versus extensive breeding (gathering and feed system) might be modified by climate changes.

• Main perceived obstacles for effective prevention and control

  Currently available antimicrobials are facing resistance in M. bovis (except fluoroquinolones, not recommended because of their importance for humans). However, development of antimicrobials suffers from a bad image regarding social responsibility.Alternatives lie mainly in vaccine development (in progress) and better herd management.Because scaling up of vaccine production is mainly feasible in pharmaceutical companies that have the production facilities, vaccine development is not very rapid. Moreover, most pharmaceutical companies are preferring that the efficacy of a potential vaccine has been demonstrated in the target species before collaborating with academic researchers and entering the final development phases.

  GAPS :

  Clinical breakpoint values need to be determined to relate in vitro AMR tests to in vivo situations. These tests should take into account the possible impact of biofilm formation. The development of resistance and mechanisms of resistance will also need investigating.

  Recommendations for “targeted” sampling (time, volume, frequency, treatments…) are needed to maximize detection (BTM, semen, etc.).

  Harmonized regulatory constraints for control of M. bovis worldwide would help in refocusing the priorities of research calls (eg semen).

• Main perceived facilitators for effective prevention and control

  M. bovis BRD is recognized as an economic disease highly ranked in the priority list of industries. Profitability is foreseen (in 5 to 10 years) for any new ATB, vaccines or diagnostics tools.

  GAPS :

  The ultimate goal (= a vaccine) seems too far to be reached (many developments are still needed). The development of a vaccine by Zoetis although imperfect is a signal of importance/ consideration of M. bovis by the industry.

Global challenges

• Antimicrobial resistance (AMR)

• Mechanism of action

  Known with a few exceptions.

  GAPS :

  Efflux mechanisms not well described.

• Conditions that reduce need for antimicrobials

  Biosecurity measures.

  GAPS :

  Need for efficient vaccines.

• Alternatives to antimicrobials

  No phage therapy. Limited work on plant derived antimicrobials.

• Impact of AMR on disease control

  Very high. Antimicrobial treatment can result in clinical recovery BUT without bacteriological clearance leading to asymptomatic carriage and shedding.

• Established links with AMR in humans

  No. These are not the same species and resistance occurs through target mutations rather than acquisition of mobile genetic elements carrying resistant genes (hence no transfer).

• Digital health

• Precision technologies available/needed

  Nothing specific for M. bovis (e.g. rumination time as an indicator of general health status in dairy, Paudyal 2021).

• Data availability

  Nothing specific for M. bovis.

• Data standardisation

  Nothing specific for M. bovis.

• Climate change

• Role of disease control for climate adaptation

  Reduction of resource use and emissions/pollution.

• Effect of disease (control) on resource use

  Less mortality implies less consumption of environmental resources to reach the same food production level.

• Effect of disease (control) on emissions and pollution (greenhouse gases, phosphate, nitrate, …)

  Less mortality implies less emissions and pollution to reach the same food production level.

• Preparedness

• Syndromic surveillance

  Not feasible as M. bovis associated clinical signs are not specific (signs overlap a.o. with BRD or mastitis). A lab diagnosis is necessary.

• Diagnostic platforms

  No need as most diagnostics are relying on commercial kits that can easily be handled by any laboratory (no requirement for expert skills).

• Mathematical modelling

  Poor data available for M. bovis. There are some studies but those based on seroprevalence (and especially those using milk as a matrix) have limitations.

  GAPS :

  Data about the reproduction number (R0) of M. bovis in different production types would be interesting. Studies investigating risk factors and good management practices resulting in reducing the BRD R0 would be welcomed.

• Intervention platforms

  Poor data available for M. bovis.

• Communication strategies

  Already lots of communication on M. bovis (website, publications, cattle associations,…).

  GAPS :

  Use other networking/communication media like twitter, tiktok.

Risk

• Impact on production.

Main critical gaps

• A better understanding of the immune response against M. bovis is needed for vaccine development.Data about routes of transmission (including through the environment and potential biofilms, semen, etc.) and infection doses per route are required to improve herd management practices.Need for experimental models for reproducing the disease in relation to development of complex cellular models.Need for clarification about the pathophysiology of M. bovis infection that could hint towards new therapeutic development.Need for interlaboratory trials to validate diagnostics methods and commercial kits. Ultimately transfer to accurate in-field tests would be most useful.Need for clinical interpretative criteria for AMR to translate MIC results in vitro into resistant, intermediary or susceptible.Need for quicker diagnostics tools including antimicrobial susceptibility testing.

Conclusion

• M. bovis associated infections are a major constraint on intensive production (both intensive beef production particularly in feed lots and milk production in high yielding herds). They have a significant negative economic impact on cattle rearing worldwide.The following factors summarise the problems: i) No effective vaccines available, ii) Insidious infection not always easily diagnosed, iii) Difficulty to eliminate the disease from a herd, iv) Difficulty to assess the contribution of M. bovis in the bovine respiratory disease complex when a number of other pathogens are also involved and finally v) Development of antibiotic resistance to most of the antibiotics currently in use.Lack of an understanding of the epidemiology of the disease at the herd level hampers the development of therapeutic preventive measures. Currently the most widely used preventive measure is chemotherapy but test and slaughter is a crude and less economical strategy to help control this disease. The disease in its chronic form should be considered also for its consequences on animal welfare (raising awareness).

Sources of information

• Expert group composition

  Florence Tardy (group leader)

  Senior specialist in animal mycoplasmoses

  Anses, Lyon

  France

  Geert Vertenten (Industry representative)

  Global Technical Director Ruminant Biologicals

  MSD Animal Health, Boxmeer

  The Netherlands

  Tarja Pohjanvirta (expert, EU)Head of section, specialist in animal infectious diseasesFinnish Food Authority, KuopioFinland

  Jose Perez-Casal (expert, Canada)Research ScientistVaccine and Infectious Disease Organization, SaskatoonCanada

  Anne Ridley (expert, EU)Lead, Mycoplasma Team,WOAH Reference Centre for Contagious AgalactiaAnimal and Plant Health Agency, Addlestone.United Kingdom

  Inna Lysnyansky (expert, Israel)Head of Mycoplasma Unit,Kimron Veterinary InstituteIsrael

  Nadeeka Wawegama (expert, Australia)Senior Research Fellow in Veterinary MicrobiologyMelbourne Veterinary School | Faculty of Veterinary and Agricultural Sciences, MelbourneAustralia

  Henk Wisselink (expert, EU)Senior scientistWageningen Bioveterinary Research, Lelystad,The Netherlands

• Reviewed by

  Project Management Board

• Date of submission by expert group

  September 1^st 2022

• References

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