Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  Some commercial ELISAs are available for detection of an antibody response to M. agalactiae. However, there are no commercially available ELISAs for mastitis if caused by the other Mycoplasma species that are also causative agents of contagious agalactia and are important in goats.

  Standard laboratory procedures are available for identifying the invading pathogen. The gold standard remains isolation of the causative organism by culture to confirm diagnosis in a country where disease is not normally present.

  A multiplex real-time PCR (Becker et al., 2012), now marketed commercially, targets all contagious agalactia agents; with the test distinguishing M. agalactiae from agents belonging to the 'Mycoplasma mycoides cluster'.

  Commercial M. agalactiae ELISA kits are available.Commercial M. agalactiae real-time PCR kits are available.For some kits, evaluation data are not published. Some kits are designed to detect (i) M. capricolum, but do not differentiate M. capricolum subspecies capricolum and M. capricolum subspecies capripneumoniae, or (ii) M. mycoides, but do not differentiate between M. mycoides subsp. mycoides and M. mycoides subsp. capri.

  List of commercially available kits (Diagnostics for Animals)

  GAPS :

  There is a requirement for fully evaluated and affordable point of care testing (pen-side tests) to ensure rapid diagnosis and rapid control measures which have been shown to perform better in stopping the disease spread.

  A reassessment of performance of currently available commercial ELISAs, through an international inter-laboratory comparison, the establishment of an international reference serum panel and /or the establishment of external quality assurance schemes for serological tests in current use would be beneficial.

  Serology for other causative agents of contagious agalactia (goats) still relies on the complement fixation test (CFT) with expertise required to perform and interpret. There is no CFT for M. putrefaciens, although this organism may be suspected from the putrid odour detectable in the milk. Improved specificity over existing tests would be needed for detecting antibody response to these other organisms, which are increasingly important in goats in Europe. This is challenging due to antigenic similarity between agents belonging to the M. mycoides cluster.

  Particular challenges with current ELISA tests include difficulties in detecting host response early in infection and in asymptomatic animals. Improved sensitivity and specificity of antibody detection tests is required.

  

• Commercial diagnostic kits available in Europe

  Antibody:There are two commercial ELISA kits for serological monitoring of M. agalactiae available in Europe. These are based on antigens comprising either a recombinant protein (P48) or a preparation of whole cells. There are no available ELISAs for other causative agents of contagious agalactia.

  Antigen:M. agalactiae: Diagnostic agar medium plates are commercially available and will assist with identification. One real-time multiplex PCR is commercially available. An international comparative study to evaluate performance would be beneficial. However, the kit which also detects, but does not differentiate between, other agents of contagious agalactia, is relatively expensive and this is likely to preclude use for many laboratories, particularly as a screening tool. Other single-plex assays are also now commercially available for M. agalactiae as well as other CA agents, but the later do not differentiate to subspecies level. A commercially developed loop-mediated isothermal amplification kit for M. agalactiae, based on the P40 gene, validated on milk samples (Tumino et al., 2020) and designed for use in the field with a portable electronic device is now available.

  Commercial M. agalactiae ELISA kits are available.Commercial M. agalactiae real-time PCR kits are available.For some kits, evaluation data are not published. Some kits are designed to detect (i) M. capricolum, but do not differentiate M. capricolum subspecies capricolum and M. capricolum subspecies capripneumoniae, or (ii) M. mycoides, but do not differentiate between M. mycoides subsp. mycoides and M. mycoides subsp. capri.

  List of commercially available kits (Diagnostics for Animals)

  GAPS :

  See Section “Commercial diagnostic kits available worldwide”.

  

• Diagnostic kits validated by International, European or National Standards

  ELISA: Only for M. agalactiae. No international standard exists, although improvements include use of reference serum previously used for other kit validations and extending performance assessment to include additional samples from different geographical regions in the world.

  ANTIGEN: Commercially available real-time PCR (M. agalactiae and the ‘M. mycoides cluster’), for which there are soon to be two manufacturers in France, was originally developed according to OIE principles for validation. Other commercial kits have recently become available.

  GAP :

  Validation data is lacking for some of diagnostic kits.

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

  OIE or EU do not stipulate. OIE makes recommendations on the choice of test types for the diagnostic purpose required.Essentially a "herd" level test the ELISA test is recommended for demonstrating that a population is free of infection, for confirming clinical cases, for contributing to eradication processes, for surveillance and determining post-vaccination response.

  Positive PCR tests should be followed up with isolation and standard methods of identification in countries or territories normally free of the disease. PCR combined by denaturing gradient gel electrophoresis (DGGE), in addition, enables identification of other Mycoplasma sp. and thus is useful for determining mixed infections.

  GAPS :

  Improved sensitivity of ELISA for detection of early stages of infection and the detection of asymptomatic carriers is required.

  A PCR restriction fragment length polymorphism method, based on the lpdA gene differentiates between M. mycoides subsp. capri and M. capricolum subsp. capricolum has been described (Cillara et al, 2015) highlights challenges associated with specific detection of other causative agents important in goats.

• Commercial potential for diagnostic kits in Europe

  ELISAs for M. agalactiae are already commercially available. For real-time PCR one multi-plex kit is now widely commercially available, with another under development, resultant increased competition may drive down costs.Sustained commercial production of the kits has a dependency on commercial viability for the producers, leading to vulnerability of supply and, for laboratories using these tests, a requirement to change to an alternative, with a different performance, requiring validation at short notice. Current ELISA diagnostic kits do not differentiate between the responses associated with vaccinated and naturally infected small ruminants. However, commercial development of a marker vaccine would drive demand for a test offering this differentiation.

  With other causative agents of contagious agalactia now predominating in goats in some areas of Southern Europe, lack of accessible commercially available serological tests may result in under-diagnosis of these other agents. Some potential exits for antibody tests identifying these other agents; however there is also a great need for accessibility and affordability of tests, thus the development of pen-side tests would be beneficial.

  GAPS:

  P48 recombinant protein, known for 20 years is in use in current commercially available ELISA. Other immunogenic proteins have subsequently been identified. One challenge is ensuring detection of infections caused by genetically and antigenically divergent strains. Ultimately, genome engineering tools derived from synthetic biology approaches are now being trialled in M. mycoides subsp. capri genome, cloned in yeast, to produce safe, attenuated strains for use in future mycoplasma vaccinology approaches (Jores et al., 2019).

  High potential for the commercial roll-out and development of easy-to-use specific and sensitive tests, with in-field test capability. The challenge is to obtain material of known provenance encompassing different stages of infection, asymptomatic infections and different countries (where strains may be highly divergent).

  Multiplexed testing for agents of small ruminant mastitis would be beneficial.

• DIVA tests required and/or available

  None available. There is not an internationally agreed approach to vaccination for contagious agalactia, with use of different commercial inactivated vaccines and inactivated autogenous vaccines permitted in Europe, while attenuated vaccines are used outside of Europe. Should a suitable vaccine become available, there will be a requirement for a DIVA test.

• Opportunities for new developments

  Further comparative whole genome sequences of M. agalactiae will enhance knowledge of epidemiology and key differences between isolates, providing a platform for post-genomic applications to identify globally conserved surface exposed antigens, expressed in the host. The organism has also been shown under experimental conditions to be able to transfer DNA between cells, indicating the potential for driving genetic diversity. An improved understanding of such mechanisms will assist the development of sensitive and specific antigen tests.

  Determination of molecular mechanisms corresponding to reduced susceptibility to therapeutic antimicrobials may also facilitate development of molecular assays to monitor susceptibility trends.

  Improvements to vectors and molecular tools have facilitated the production of fusion proteins on commercial scale.

  Lateral flow assays recently described for M. agalactiae (Remesh et al., 2014), require further evaluation and commercial development.

  Isothermal amplification assays, such as LAMP and RPA for direct detection of the M. agalactiae antigen in clinical samples on the farm have been developed (Rekha et al., 2015), and commercially developed in Italy (Tumino et al., 2020). Assay targets must be tested against a diverse range of strains and the appropriate range of sample types to ensure sensitive and specific detection. Assuming benefits can be realised for M. agalactiae, similar penside tests for other causative agents of contagious agalactia would also be beneficial.A multiplex PCR test is available for M. agalactiae, differentiating this from other contagious agalactia agents. It has been used to screen bulk tank milk samples in France (Tardy et al., 2019). Determination of appropriate cut-off values for naturally infected samples is challenging, and would benefit from an international ring trial. However, currently the test is expensive, which is likely to limit widespread use for monitoring purposes. Efficiency of the DNA extraction method, and particularly the ability to remove inhibitors from complex matrices, is an important consideration.

  GAPS :

  Antibody detection assays to include improved ELISA, lateral flow, latex agglutination tests should be assessed for performance in detecting earliest response to infection, and to that associated with chronically infected / asymptomatic carriers.

  Identification of specific markers of infection in the host and development of novel approaches to detect these.

  Development and /or commercial availablity of penside tests for antibody and antigen detection.

  Insight of genomic and antigenic divergence globally, using representative strain sets; leading to improved knowledge and understanding of events shaping diversity.

  Formalised scheme to characterise and track strains (e.g. core genome MLST) to help improve understanding of global epidemiology.

  Advanced knowledge of the organism’s metabolic requirements and host adaptation will facilitate development of more specific culture media.

  External Quality Assurance schemes for tests to detect M. agalactiae, as well as other causative organisms of contagious agalactia, would be beneficial.

• Vaccines availability

• Commercial vaccines availability (globally)

  A variety of vaccines aimed at preventing contagious agalactia due to M. agalactiae are produced and used widely in the Mediterranean countries of Europe and in western Asia. The majority are inactivated whole cell vaccines.

  Live vaccines are not permitted in Europe but inactivated, adjuvanted vaccines are available commercially, with inactivated autogenous vaccines also used. However, due to decreasing antibody titre as determined by available antibody detection tests, vaccination needs to be repeated at 4-6 monthly intervals (depending on product), which is expensive and often impractical. Outside of Europe, live attenuated vaccines may be used. Comparative experimental studies indicate that live attenuated vaccines, such as that used in Turkey, have greater efficacy than inactivated vaccines in experimental challenge trials. Note: Some countries free of contagious agalactia will use a slaughter policy to control disease incursions, so vaccination is not permitted.

  For goats, dual, or multiplex vaccines against M. agalactiae and M. mycoides subsp. capri with some also including M. capricolum subsp. capricolum are available and developed commercially. A multivalent formalin inactivated vaccine incorporating all four causative mycoplasmas was trialled experimentally and despite showing some promise (in preventing new clinical signs), was not developed commercially.

  GAP :

  Published, peer reviewed, efficacy data for use in the field is still lacking.

• Commercial vaccines authorised in Europe

  In Europe formalin inactivated, adjuvanted vaccines are available commercially. Aside from experimental studies, either comparing available commercial products, or inactivated versus live (attenuated Turkish) vaccine, the efficacy of these inactivated vaccines and the need to revaccinate frequently remains a concern. A recent experimental study confirmed efficacy of a saponinized inactivated vaccine against M. agalactiae produced high levels of immunoglobulin, and was protective (Ozdemir et al., 2019), warranting further exploration.

  GAP :

  Vaccine efficacy data, particularly where used in the field in Europe is required.

• Marker vaccines available worldwide

  Currently there are no marker vaccines available.

• Marker vaccines authorised in Europe

  Currently there are no marker vaccines available.

• Effectiveness of vaccines / Main shortcomings of current vaccines

  Inactivated and live attenuated vaccines are available. However, there is lack of consensus regarding the effectiveness of the (commercially available) inactivated vaccines. M. agalactiae-only formulations may be unsuitable in goats where the other causative organisms may be causing disease and this has largely been addressed by commercial manufacturers, with multi-organism formulations available commercially. Protection is, however, short-term with re-vaccination recommended every 4-6 months. A number of countries, including those in the EU, do not allow the use of live vaccines. While live vaccines have been shown to be more effective in trials (and in some countries, used for many years) lack of serological response is noted, but this does not appear to hamper protection.

  Reports on use of autogenous vaccines are scarce, with little data to support effectiveness, through follow up monitoring. These should be considered a useful addition, or feasible substitutes to licensed vaccines, but only bring local solutions. Protocols for use of such vaccines requires harmonisation.

  GAPS :

  Improved knowledge of the mechanism conferring the attenuation, and the stability, of currently available and future attenuated vaccine strains is required.

  Knowledge of efficacy of attenuated vaccines in males and non-lactating females, for the control of disease is also required.

  Effect of different adjuvants, combined with best concentration of targeted antigen, requires investigation.

• Commercial potential for vaccines in Europe

  There is potential in some European countries for vaccination. The development of a safe, preferably marker, vaccine which provides greater and longer lasting protection is much needed. Although live vaccines are banned in Europe, current experimental work is focused on development of next generation fully attenuated synthetic vaccines for Mycoplasma diseases. There could be significant commercial potential for marker vaccines of proven efficacy and stability in European countries in which contagious agalactia is present.

  Genomic diversity of M. agalactiae strains, which is becoming better understood, should be utilised to enable development of marker vaccines, and, correspondingly, tests able to differentiate between field and vaccine strains, as current tools lack the ability to differentiate the serological response.

  There could be significant commercial potential for single-shot marker vaccines, with robustly proven efficacy in European countries in which contagious agalactia caused by M. agalactiae is present. Any vaccine developed should be efficacious against all strains, stable, long lasting protection from a single shot, be effective against all clinical signs, able to be administrated in males and non-lactating females and suitable for use in all countries in which the disease occurs.

  Although M. agalactiae is most important in ovines, M. mycoides subsp. capri, in particular, is important in goats in some European countries, or regions therein. Complete attenuation of a M. mycoides subsp. capri strain using a synthetic genomics approach has already been achieved experimentally (Jores et al., 2019), thus there will be substantial interest in further development of this approach.

• Regulatory and/or policy challenges to approval

  For those countries that have eradicated the disease, or where the disease does not currently occur, there will be policy challenges for approval.

• Commercial feasibility (e.g manufacturing)

  This would dependent on what is developed, for example use of genetically modified vaccines could face resistance in some countries, with concerns regarding release of genetically modified organisms (GMOs) to the environment, despite the supposed fragility of mycoplasmas to environmental stress. Moreover, the financial return for investment in a product for sheep and goats may limit commercial development of such vaccines, particularly for other contagious agalactia agents.

• Opportunity for barrier protection

  No. A vaccine capable of protecting livestock in endemic zones, able to be used on all sheep and goats and without risk of excretion by lactating animals, or onward transmission, is required.

• Opportunity for new developments

  Ultimately genomic and functional analysis studies to identify conserved repertoire of surface exposed or secreted putative immunogens, by comparing a diverse range of strains, should determine feasibility of consensus species-specific target genes, or suitable targets for dual vaccine for protection against the other disease agents.

  Difficulties in accessing funding to develop new vaccines may be a barrier, despite economic importance of disease in countries experiencing endemicity, with challenges also faced trying to eradicate, or otherwise control, the disease.

  GAPS :

  A more efficacious and long lasting vaccine able to cover infection by diverse M. agalactiae strains and suitable for use globally needs developing. A DIVA would be beneficial.

  Efforts are required for global standardisation of vaccine efficacy assessment requirements and to harmonize regulatory constraints worldwide for vaccine use.

  Lack of harmonised requirements for the manufacture and control of autogenous vaccines across the EU. Monitoring the effectiveness of these vaccines, where used would be beneficial.

  Standardised infection models, with consideration for different clinical manifestation of disease would be beneficial.

• Pharmaceutical availability

• Current therapy (curative and preventive)

  Formal restriction and slaughter policies are statutory in many countries or territories that are normally free of contagious agalactia.Antibiotics are used in many countries where the disease is endemic. Lacking a cell wall limits the choice of antibiotics, with comparatively few antibiotics licenced for small ruminants. Tetracyclines, macrolides are used, with fluoroquinolones also effective. Phenicols (florfenicol), pleuromutilins (tiamulin) and aminosides now less commonly used. Although the mycoplasmastatic antibiotics are effective in reducing the severity of clinical signs they may not eliminate the organism. Fluoroquinolones are recommended only when there are no alternative antimicrobials authorised for the respective target species and indications. Disinfection and fly/ tick control are appropriate as adjuncts, as should milking machine maintenance and cleanliness of other equipment.Reduced susceptibility to some macrolides, tetracyclines, lincosamides, phenicols and, rarely, to third generation fluoroquinolones have been reported for field isolates from some countries, but MIC values are generally low in the populations studied. For other agents of contagious agalactia, MIC values reported in field isolates have generally been lower than found for M. agalactiae.Mutations in 16S rRNA (tetracyclines) and 23S rRNA (macrolides) genes reported to confer reduced susceptibility, but some changes are not accounted for suggesting other, as yet unidentified (such as efflux), mechanisms exist. Mutations in the quinolone resistance determining region confer reduced susceptibility to the newer fluoroquinolones.

  GAP :

  Lack of standardised laboratory methods, control strains and interpretive criteria for minimum inhibitory concentration (MIC) testing, with no clinical breakpoints, or epidemiological cut-off values, available to facilitate data interpretation, comparison between countries and correlation of MICs with in vivo efficacy.

• Future therapy

  In the short term, therapy is likely to remain with the use of antibiotics, with or without vaccination, in combination. More effective, targeted, anti-mycoplasma therapeutics are required. However, the initiatives to drive down antimicrobial use in the EU is combined with lack of new antibiotics under development.Assessment of activity of plant-based, and other naturally occurring, antimicrobial alternatives is in its infancy, with few studies yet to include M. agalactiae. Limited recent experimental studies, including other causative agents of contagious agalactia, show early promise (Kami-Kami, 2017).

  GAP :

  The relatively high MIC values reported suggest that a key concern surrounding naturally occurring antimicrobials will be determining whether they can offer a practical and safe alternative to antibiotics. Harmonisation of procedures used to demonstrate efficacy would be beneficial to facilitate data comparisons.

• Commercial potential for pharmaceuticals in Europe

  High potential in those countries affected, but with the driver for reducing antimicrobial use in Europe there could be constraints to applications, in view of concerns regarding resistance development. The development of alternative strategies for control of contagious agalactia, including more effective vaccines, is warranted.

• Regulatory and/or policy challenges to approval

  None anticipated, other than the driver to reduce use, although consideration of meat and milk withdrawal times will be important.

• Commercial feasibility (e.g manufacturing)

  Should be commercially feasible, although dependency on market, cost, withdrawal times, policy drivers and demand. This also applies to other causative agents of contagious agalactia.

• Opportunities for new developments

  Identification of as yet unknown mechanisms associated with reduced susceptibility to key therapeutic antibiotics, including a possible role for transporter molecules and horizontal gene transfer.

  Chromosomal horizontal transmission and genome reshuffling has been demonstrated using isogenic mutants under enrofloxacin stress in laboratory experiments (Faucher al., 2019). Whether this may also occur under different antibiotic stressors and in the host remains to be determined.

  Improving knowledge of resistance mutation frequency and stability under antibiotic pressure in field, compared with laboratory isolates, for development of rapid identification.

  Genome and post-genomic transcriptomic, metabolomic and immunoproteome exploration tools can be used to identify potential target transporter (and other) functional proteins of relevance.

  GAPS :

  More effective, targeted, treatment strategies that minimise the excretion of antibiotics into the environment are required.

  Paucity of data for evaluating the future potential of plant or other natural antimicrobial compounds.

  Availability of a standardised approach to correlate MIC to epidemiological cut-offs, or breakpoints corresponding to resistance mechanisms, for clinically relevant antimicrobials.

• New developments for diagnostic tests

• Requirements for diagnostics development

  Rapid, cost effective, easy to use and interpret.Evaluation of performance of current tests, with comparisons between EU and "third" countries.Agree standardised controls and harmonisation of approaches.Commercial development and independent validation of high-performance antibody-based detection tests for early and chronic infection stages.Development, field evaluation and commercial marketing (as appropriate) of field applicable diagnostic tests for rapid identification of infected sheep and goats.One-step contagious agalactia syndrome diagnostic tests suitable for field use would be beneficial for use with goat populations.Suitable for individual animal and herd level diagnosis and surveillance.Quality controlled/standardised tests, with regular proficiency testing schemes for testing laboratories.

  GAPS :

  A major challenge is the identification and interpretation of asymptomatic carriers.

  Understanding of whether disease resistance exists for contagious agalactia and link to resistance to other diseases of small ruminants.

  Further knowledge of the dynamics of host response and pathogen shedding variability associated with false negative serological test outcomes is required.

  Development of easy to use and cheap pen-side tests for testing milk/sera would improve diagnosis. A new commercial isothermal LAMP test, based on the P40 gene, has now been released, warranting inter-laboratory assessment for rapid and accurate detection of genetically divergent strains. Roll-out of such tests should help increase awareness of this disease and thus improve prevalence knowledge.

• Time to develop new or improved diagnostics

  Although development of diagnostic tests is more rapid, less rigorous and less expensive than for vaccine or antimicrobial development, it is a major commitment to develop and evaluate diagnostic tests, using the best available proven negative and proven positive test material, so the assessed demand for end product must be high for return in investment. In relation to a proposed marker vaccines the diagnostic kits should be developed in parallel.

• Cost of developing new or improved diagnostics and their validation

  This could be significant and a careful assessment of the expected market is required for commercial interest. Developing new tests requires investment of time, development of collaborations to source the most appropriate test materials and consumables, production of individual components and reagents, assessment of intra-, as well as inter-laboratory robustness to validate performance and the reading, and interpretation, of test results.

• Research requirements for new or improved diagnostics

  Further knowledge of the sequence of events following initial attachment of the organism. Includes the interplay of the variable, surface exposed, M. agalactiae surface variable lipoproteins (vpmas) with other putative virulence candidates associated with invasion, evasion of host response and microbial dissemination with evaluation in vivo.Improved understanding of response of the ovine and caprine host to initial infection, with identification of any organism specific markers of infection in the host.Improved understanding of tropism for mammary tissue and associated lymph nodes, and understanding as to why (and the mechanisms behind) the organism colonises and invades other body sites. Understanding of the genomic and antigenic diversity of M. agalactiae population through sequencing of strains from different global locations and associated with different (including neurological) clinical signs, to ensure diagnostic tests detect infections by genetically diverse strains. This would also be beneficial for other CA agents.

• Technology to determine virus freedom in animals

  Improvements to sensitivity of serological tests, including ability to detect antibody response in chronically infected / carrier animals and rapid, robust and specific molecular tests that can be applied on-site in developing countries.Improved surveillance strategies: establishment of pre-export, as well as post import, testing. Rigorous DNA-based testing of germplasm, with serological testing of rams and bucks used for mating.Clearly defined risk-based sampling and testing requirements for demonstrating disease freedom.Understand drivers for genetic diversity and characterisation of isolates to ensure strong, or develop improved, coverage of new types by existing tests.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  Standardisation of procedures for preparation and validation. Use of saponized inactivated vaccine against M. agalactiae have been found to offer better protection and immune response, than formalin, heat and sodium hypochlorite inactivated products.Improved knowledge of host response to lengthen period of immunity.Improved knowledge of immunogenic effects of different adjuvants, or alternative synthetic vaccine delivery systems.Research in vaccinology needs to emphasise the development of marker vaccines that are both efficacious and permit ready analysis of vaccinated, as opposed to infected, animals. Spread of the disease to, and establishment in, other countries currently not allowing vaccination would mean it becomes imperative to distinguish vaccinated from naturally infected animals in order to allow use of such vaccines.Suitable for use in lactating animals, males and pregnant females.Effective against all strains.

  GAPS :

  Much current research is focused on identifying immunogenic proteins that can be utilized in subunit vaccines. In addition, recent developments in epitope mapping, combined with whole genome sequence analysis, should facilitate identification immunogenic proteins that may be suitable candidates in vaccine development. Strongly immunogenic lipoproteins have been identified in M. agalactiae as judged by reaction of antibodies in the sera of infected sheep.

  Antigenic variation, affecting stability of some of these immunogenic proteins, hinders applicability as a suitable marker. However, other recently identified proteins, as well as P48, or AvgC, may be of value in future subunit vaccines.

  Greater understanding of the impact of antigenic variability and rapid switching of expression of the variable surface lipoproteins, vpmas, which has been demonstrated in vivo (Baranowski et al., 2014), on protection against challenge.

• Time to develop new or improved vaccines

  This will be significant, irrespective of whether it applies to M. agalactiae, or other causative agents of contagious agalactia. A candidate vaccine first needs to be identified. The design, protocol approval and clinical trials process including collection of the data necessary to obtain the necessary licencing will take some years. The registration process in Europe would take a minimum of one year. Moreover, the development and approval of animal disease infection models for both sheep and goats for all Mycoplasma-associated contagious agalactia organisms suitable for testing vaccines is required.

• Cost of developing new or improved vaccines and their validation

  This is liable to be expensive; there would be the need to develop the product and undertake the necessary testing requirements for licencing. Since the disease is often prevalent in countries with a lower socio-economic structure, the potential for recuperation of the investment would need careful consideration. Challenges to setting up field trial/s. For the causative organisms associated with goats, the contribution of each species is unknown in many parts of the world, so assessing likely recuperation is challenging. If a company wanted to carry out vaccine development work in a country free of contagious agalactia, then regulations may impose registration and containment facility requirements.

• Research requirements for new or improved vaccines

  Improved knowledge of causative organism(s): characterisation of conserved and surface exposed immunogenic proteins likely to induce protective immune response.Improved knowledge of the response of the host to the infection process.Identification of candidate antigens, should make use whole genome data from a range of diverse strains with accompanying functional, bioinformatic, proteomic, and biological information.Improved knowledge of the immuno-stimulatory effects of different adjuvant formulations and, in future, synthetic delivery systems.There are good models for the mastitis forms of disease, but other models (arthritis, keratoconjuctivitis and, for other contagious agalactia agents, respiratory) are less established.There is a need to assess ways to improve of the immune protection of the target organ (udder) as it is estimated that in approximately 90% of cases, M. agalactiae enters the host via the teat canal. This could include consideration of alternative administration routes, such as vaccination of the organ, via the intra-canalicular route.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  There may be some potential for antibiotics with increased efficacy/distribution that could prevent the continued excretion of mycoplasma, or at least minimise the period that antibiotics are excreted. However, this would need to be established through controlled experiments.Standardized laboratory methods and interpretive processes for MIC testing of veterinary mycoplasmas that may be extrapolated to other antimicrobial compounds, including plant extracts, to facilitate data interpretation and correlation of MICs between studies and in future studies of in vivo efficacy.Minimise contamination of the environment by improving mycoplasmacidal effects of the products used to clean the animal.

  GAPS :

  A single study in vivo showed promise of an un-named bioproduct in reducing clinical symptoms and, within the study period, reportedly prevented recurrence of clinical signs in the treated animals (Silva et al., 2013). Further comparative studies are warranted, including an extended period of monitoring to determine whether the organism has been eliminated and asymptomatic carriage state avoided. Although anti-mycoplasma activity of locally sourced plant extracts have been investigated, mainly for the other disease agents (Al Momani et al 2007; Kama-Kama et al, 2016, 2017; Shah et al, 2017; Lopez et al, 2013) in vivo efficacy, with additional considerations for bioavailability and safety of such extracts, are yet to be established.

  Although many single polymorphic nucleotide changes associated with reduced susceptibility have been decoded for several of the key therapeutic antibiotics, knowledge of other mechanisms of resistance, for example importance of efflux and potential for horizontal transfer, need investigating.

  

• Time to develop new or improved pharmaceuticals

  Expected be significant. The time to develop would depend on the product and the trials required to validate efficacy and safety, with together with collection and assessment of the data necessary to gain approval is likely to be years. Commercial production requires additional time, with the registration process in Europe at least a further year. Minimum of five (to ten) years would be anticipated.

• Cost of developing new or improved pharmaceuticals and their validation

  This is also liable to be significant, but is difficult to assess as there is dependency on the type of product and the trials required for assessment and validation. A further challenge is that contagious agalactia is often present in countries with a generally lower socio-economic structure and the potential for recovery of investment would need careful consideration.

• Research requirements for new or improved pharmaceuticals

  Consideration of mechanisms conferring reduced susceptibility / resistance not already established.Optimisation of dosing regimen: pharmacokinetic/pharmacodynamic parameters.Considerations of withdrawal time.Consideration of synergism/antagonistic properties to reduce need for high concentrations with plant based bio-products.Optimisation of extraction and storage procedures applied to plant based bio-products.

  GAPS :

  Laboratory methods and interpretive criteria for minimal inhibitory and mycoplasmacidal testing needs to be agreed and standardised, including use of standardised control strains and prioritised list of antimicrobial agents and concentrations.

  Clinical breakpoints ultimately need to be established to enable correlation of MICs with in vivo efficacy.

Disease details

• Description and characteristics

• Pathogen

  Mycoplasma agalactiae has traditionally been considered the main pathogen in sheep and goats. However, Mycoplasma capricolum subsp. capricolum and Mycoplasma mycoides subsp. capri (formerly M. mycoides subsp. mycoides biotype Large Colony, Manso-Silvan et al, 2009) are particularly important in goats, along with Mycoplasma putrefaciens, which causes mastitis and arthritis. Mixed infections involving several Mycoplasma species have also been reported in symptomatic and asymptomatic (Gil et al, 2003; de la Fe, 2005; de la Fe et al, 2009) goat herds, with the balance between M. agalactiae and other members of the M. mycoides cluster infection highly region-dependent.

  It should be noted that M. agalactiae shares high biochemical and genetic similarity with Mycoplasma bovis a pathogen of bovines, which has occasionally been isolated from sheep and goats.

  GAP :

  Data collected by OIE does not distinguish between the causative pathogens. Greater focus on identification of other agents of contagious agalactia in goats is required, but the lack of highly specific, easy to use diagnostic tests presents challenges.

• Variability of the disease

  Mastitis, arthritis and keratoconjunctivitis can occur in both male and female sheep and goats. The disease presents itself differently in sheep and goats. The major pathogen(M. agalactiae) was considered to be unusually homogenous, with dominant subtype that has disseminated within and between countries. However, in other areas genetically divergent strains exist, defining the geographical location of outbreaks. Contagious agalactia in sheep generally presents as a less severe disease than in goats and is caused almost always caused by M. agalactiae. Clinical disease can be manifested in an acute, sub-acute or chronic form. During fulminating outbreaks in areas previously free from the disease, the initial stages of the acute form of contagious agalactia may involve septicaemia and a febrile illness. Where M. agalactiae is enzootic, a discrete disease is usual which requires differentiation from other (including viral, bacterial, other mycoplasmal causes). A sporadic occurrence of atypical, or asymptomatic, forms has also been reported. The organism has also been found in large quantities in the brain, where it may be responsible for non-purulent encephalitis as well as ataxia in young animals.Goats are also affected by M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens and, in general, disease signs are more pronounced in goats; disease is also more likely to be acute, or even hyper-acute, with severe respiratory signs and higher morbidity and mortality. In particular, M. mycoides subsp. capri can also cause a pleuropneumonia in goats, with clinical signs indistinguishable from those caused by contagious caprine pleuropneumonia, a disease of small ruminants found in Africa and Asia. M. putrefaciens is more likely to be associated with a sudden drop in milk production without other clinical signs. M. capricolum subsp. capricolum is still rare in Europe, but together with M. mycoides subsp. capri and M. putrefaciens has overtaken M. agalactiae as identified causative agents of the disease in goats in France.

  M. mycoides subsp. capri isolated from clinically healthy as well as diseased goats and has been recovered from the brains of asymptomatic auricular carriers. This organism appears to persist, including in heathy sheep, in Australia, despite absence of any associated disease in that country. Multiple species infections have also been reported (Al-Momani, et al., 2006).

  GAPS :

  Subclinical CA is one of the main problem regarding transmission risk: rapid (but sensitive) lab detection is essential

  The role of pneumonia as part of the infectious / transmission process for M. agalactiae has not been clearly defined. Furthermore the association of the presence of M. agalactiae (and M. mycoides subsp. capri) in the brain, with ataxia seen in newly born lambs and kids.

  Outbreaks of severe polyarthritis have been associated with M. mycoides subsp. capri in kids with little effect on adults.

  M putrefaciens is more likely to be associated with a sudden drop in milk production without other clinical signs.

  Evidence for pathogenic strains still anecdotal and requires animal infections studies. So called apathogenic strain of M. mycoides subsp. capri still capable of causing death and disease.

  Contribution of horizontal gene transfer (and role of mycoplasma integrated conjugative elements (ICE) to M. agalactiae strain evolution of are required, contrasting with clonal dissemination of a dominant subtype over many years. Strain characterisation remains challenging within enzootic areas for outbreak tracing.

• Stability of the agent/pathogen in the environment

  Mycoplasmas can survive for one or two weeks at room temperature but are sensitive to osmotic shock, the effect of detergents, disinfectants and ultraviolet radiation in laboratory studies. M. agalactiae has been recovered from farm environmental matrices. Survival of M. agalactiae is prolonged in farm matrices, particularly at lower temperatures (20^oC / 4^oC). Of the causative agents of contagious agalactia, all four have appear produce biofilms in vitro under laboratory experimental conditions (unpublished data). Biofilm grown cells were more resistant to stress, including heat and desiccation. However, whether the ability to produce biofilms is shared by all strains remains to be determined.

  GAP :

  Biofilms are believed to contribute to persistence in the environment and may account for “mal di sito”, first reported over 100 years ago, whereby flocks could become infected from a contaminated environment.Improved understanding regarding the occurrence, and role, of biofilms in survival in the environment, and on milking equipment, is required.The metabolic and virulence properties of biofilm grown, compared with planktonic, strains remain to be determined.The presence of organic matrices is known to adversely affect disinfectant efficacy for other organisms, thus an improved understanding of impact of organic matrices on disinfectant efficacy is required.

• Species involved

• Animal infected/carrier/disease

  Sheep and goats are the primary species involved. Clinical disease (pneumonia with, or without, keratoconjunctivitis) has been reported in the Alpine Ibex and chamoix, and keratoconjuctivis in iberian ibex. Other animal species, such as camels, cattle, or small wild ruminants (deer, ibexes) can function as infection reservoirs. Antibodies have also been detected in South American camelids, (Llamas, Alpacas, Vicunas), but recovery of the organism from such animals has not, to date, been reported.

  GAPS :

  Precise role of domestic and wild small ruminants (e.g. ungulates) as reservoirs is required for the development of risk-based surveillance approaches.

  Role of other common European wild animals (mammalians, birds) as reservoir in enzootic areas is also unknown.

  As contagious agalactia causing mycoplasmas have never been isolated from camelids, further work is required to verify camelid infection and determine importance as a reservoir.

  Precise role of asymptomatic carriers in disease transmission remains to be demonstrated experimentally.

• Human infected/disease

  M. agalactiae is currently not thought to be pathogenic to humans.

  GAPS :

  No evidence of zoonosis from CA mycoplasmas though unconfirmed reports of sickness in humans working with calves fed goat milk naturally affected with M. m. capri (Goncalves 2009).

  Mycoplasma capricolum subsp. capricolum was isolated from a case of septicaemia in a hospitalised immunocompromised patient, but source of origin, or evidence of contact with potentially infected animals, could not be identified (Heller et al, 2015).

• Vector cyclical/non-cyclical

  Ticks, mites, or other biting insects, may have a role in transmission of this disease, but this has not been demonstrated experimentally.Milking equipment and the hands of milkers have an important role in transmission.

  GAP :

  Further work is needed to confirm whether insects and ticks (often present in the ears) are capable of infecting the small ruminant host rather than just carrying the mycoplasmas.

• Reservoir (animal, environment)

  Other domestic and wild ungulates; M. agalactiae has been isolated from cattle. Whether other domestic animals or wildlife can act as a reservoir has not specifically been targeted for study.The environment of a small ruminant farm can act as a reservoir. Dissemination into the environment occurs by means of ocular and nasal discharge, milk, faeces, urine, abortifacient material, and excretions from open joints or the male genitourinary tract.

  Asymptomatic carriers in a herd/flock pose a serious risk.

  GAPS :

  Identification of key environmental reservoirs and demonstration of environmental survival and persistence in the farm environment. However, proving transmission from the environment will be challenging.

  Although presence of viable organisms in urine and faeces has been demonstrated, there is little evidence to indicate transmission of mycoplasma via urine and faeces, compared to milk, and work is needed to assess the importance of excreta in transmission.

  Identification of key environmental reservoirs and demonstration of environmental survival and persistence in the farm environment. However, proving transmission from the environment will be challenging.

  Although presence of viable organisms in urine and faeces has been demonstrated, there is little evidence to indicate transmission of mycoplasma via urine and faeces, compared to milk, and work is needed to assess the importance of excreta in transmission.

• Description of infection & disease in natural hosts

• Transmissibility

  Horizontal transmission occurs by contact between infected animals and/or the environment. Animals become infected by ingestion or occasionally by inhalation. Aerosol transmission is possible over short distances. Transmission via fomites, (milking equipment or milker’s hands) is possible, with a manual hand milking challenge model of infection able to induce disease. Vertical transmission can occur by means of suckling milk from infected mothers. Rams and bucks used in mating and for artificial insemination can act as asymptomatic carriers, with viable organisms present in semen/ germplasm (Gomez-Martin et al., 2015).

  GAPS :

  Vertical transmission can also occur through the transplacental route and give birth to kids and lambs with swollen joints or cause abortions.

  In utero transmission for kids is described, but the incidence of ante-partum and per-partum transmission in lambs still requires attention.

  The importance of the organism’s ability to rapidly switch the expression of surface exposed variable antigens to evade the immune system in vivo has now been demonstrated. However, further investigation is required of the immunity effectors and pathways, particularly for lambs not experiencing disease, and with regards to influencing of patterns of milk shedding within persistently infected flocks.

  Risks of transmission through venereal transmission and infected germplasm (Wrathall et al., 2007) requires further investigation.

  

• Pathogenic life cycle stages

  Experimental focus has been on M. agalactiae which has been shown to spread systemically during experimental intra-mammary infection of lactating ewes. This appears to be multifactorial, involving several genomic loci and surface exposed membrane proteins, including the phase-variable surface lipoproteins (vpmas). These have recently been demonstrated through to play important roles in adhesion to and translocation through host cells and evasion of the host immune system (Baranowski et al, 2014; Czurda et al, 2017; Hegde et al, 2018).

  Development of chronic and sporadic asymptomatic conditions has also been reproduced experimentally and is also associated with the capacity of M. agalactiae to evade the host response.

  Genetically indistinguishable isolates using a suppression subtractive hybridization approach of M. mycoides subsp. capri recovered from both septicaemic asymptomatic goats in the field appear to have equal capacity to cause death following experimental challenge of naïve goats (Tardy et al, 2011).

  GAPS :

  Further understanding of the host/pathogen interplay, including the function of other immunogenic proteins and identified polysaccharides in virulence.

  Factors influencing tissue and mucosal surface tropism (and, for other agents of contagious agalactia, the respiratory system) and virulence associated with other, or unusual, clinical signs.

  Factors promoting immune tolerance or strain dormancy associated with asymptomatic carriage, compared with clearance of infection.

  

• Signs/Morbidity

  Most common disease presentation associated with infection by M. agalactiae is chronic, in both sheep and goats. Acute and sub-acute episodes do occur with M. agalactiae and have been more frequently reported in goats. However, in general the hyper-acute and acute forms of the disease are more commonly associated with M. mycoides subsp. capri, M. capricolum subsp. capricolum or M. putrefaciens affecting goats and often with respiratory complications. Acute infections begin with a transient fever followed by malaise, inappetance and mastitis. The udder becomes hot and swollen and the milk is usually greenish-yellow or greyish-blue and watery at first before becoming lumpy. Polyarthritis is also common especially in the tarsal and carpal joints and this may be the major clinical sign in male goats; this clinical sign can affect the efficiency of mating by male goats or rams. Keratoconjunctivitis develops in approximately half of all infections and is usually transient but may become chronic. Animals can occasionally become blind in one, or both, eyes. Abortions can occur in chronically infected animals. Ataxia may be observed in newborns.

  GAPS :

  Generally arthritis and septicaemia in kids is not a feature of M. agalactiae, but instead, of M. mycoides subsp. capri without clinically affecting adults. The host-pathogen factors affecting this, require investigation to determine whether these differences are truly reflective of the aetiological-agent.

  Often very little ocular involvement in M. mycoides subsp. capri infections and the reasons for this are unclear.

  Some disagreement about relative severity of disease in small ruminants with sheep in southern Italy presenting more severe signs from infection with M. agalactiae.

  Consideration for welfare/ disease susceptibility associated with breed is warranted.

  The link between M. agalactiae found in the brain to ataxia and non-purulent encephalitis in young animals remains to be established.

• Incubation period

  The incubation period may last from one week to two or more months, with duration being related to the degree of virulence of the infectious agent and the overall resistance by the host immune system.

  GAPS :

  Very long incubation periods occur, for at least some strains, with cases appearing gradually. In some cases infection is sporadic. Factors associated with reactivation of disease signs are not well known, although females appear vulnerable at onset of lactation.

  The incubation period can be difficult to define, owing to clinical signs not necessarily being pathognomic and challenges associated with determining the time of infection. Experimentally, incubation for mastitis is a few days. However, in the field this is affected by immune status, age, health of, and stress in, the host, with poor management systems, animal movements and onset of lactation also having likely roles in the speed of disease development.

• Mortality

  When an organism is first introduced into a susceptible flock or herd the morbidity rate is approximately 30 – 60% and the mortality rate is usually less than 20% in adults and lactating animals, although can be higher, up to 40% in goats. However, it may reach 40 – 70% in young, often nursing, lambs or kids with septicaemia. Severe forms may occur more frequently following infection with contagious agalactia agents belong to the M. mycoides cluster; in goats the mortality rate following infection with M. mycoides subsp. capri or M. capricolum subsp. capricolum is typically higher than for M. agalactiae (up to 100%), particularly in young animals.

  GAP :

  Factors influencing morbidity/mortality ratios, which vary substantially, are not clear.

• Shedding kinetic patterns

  The infectious organism is shed during infection. Following alleviation of clinical signs of the disease the causal agent is excreted with milk for several months, up to maximum of 8 years, although the excretion pattern is often intermittent. Asymptomatic carriers (sheep and goats) may harbour the infectious agent in their genital tracts and in males the organism may be excreted in semen. In goats it is thought the infection can be present in the external auditory canal, although the reliability of ear swabbing for identifying infected animals is debated.

  GAPS :

  Excretion from eyes and nose is very transient, 1-2 weeks, with likely role in transmission through contact and aerosols.

  The role faecal and urinary tract shedding of M. agalactiae in transmission should be investigated.

  Factors affecting shedding patterns in chronic and latent infection states.

  Demonstration of role of asymptomatic carriage, with intermittent shedding, in transmission.

• Mechanism of pathogenicity

  Lacking much of the machinery known to mediate virulence in other organisms, the relationship of M. agalactiae with its host and the response of the host following colonisation appears to play a major role in infection. In animals infected orally it is assumed that the primary site of adhesion, and subsequent invasion is the small intestine. Infection via the mammary gland, thought to be via defective or poorly cleaned milking equipment. Mycoplasma lipoproteins appear to be key to inducing an adaptive host response and immune evasion enabling dissemination within the infected host. In M. agalactiae the surface exposed vpma lipoproteins, have a pivotal role in the subsequent adhesion, invasion and dissemination from the mammary gland. Evidence is also accumulating for a role for polysaccharides in virulence. Evasion of the immune system appears to be a key strategy used by the organism to facilitate its own survival in the host environment. Infected animals may develop bacteraemia and the infectious agent is transferred via the blood to the target organs.

  GAPS :

  Although recently demonstrated to have a significant influence on the pathogenicity, through immune evasion, adhesion and invasion, the exact role of the important phase-variable vpma surface proteins in the interplay between the organism and the host remains to be determined.

  The role of other surface exposed/excreted immunogens, their interplay with the vpma proteins, and in particular their role of invasion and of lymphatic and intracellular dissemination also requires investigation.

  Although enteric colonisation is assumed and the organism is shed in faeces there is little evidence for intestinal route of infection. While considered more likely to be invasion of lymph glands/palatal tonsils, this remains a clear knowledge gap.

  The contribution of the host immune response to lesion development in the mammary gland (and for M. mycoides subsp. capri in particular, lung) and to keratoconjunctivitis requires investigation.

  Whether there is a similar role for variable and other surface exposed proteins in adhesion and immune evasion in the pathogenesis of other agents of contagious agalactia requires investigation.

• Zoonotic potential

• Reported incidence in humans

  There is no evidence to date that M. agalactiae is a threat to human health.

  GAP :

  See Section “Species involved - Human infected/disease.

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

  N/A.

• Symptoms described in humans

  N/A.

• Estimated level of under-reporting in humans

  N/A.

  GAP :

  M. agalactiae, or any of the other causative organisms of this disease, would not normally be considered.

• Likelihood of spread in humans

  N/A.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Serious impact on animal welfare. Contagious agalactia can also result in serious economic losses associated with treatment, deaths, abortions, lowered milk production, spoiled products and general reductions in the productivity of livestock. Good herd management with biosecurity measures is important for prevention. Control in countries where the disease is endemic is by vaccination and the use of antibiotics, although while these alleviate clinical signs, microbiological elimination is frequently not achieved as key therapeutic antimicrobials in used in Europe are mycoplasmastatic, often leaving treated animals as carriers. Treatment may also reduce the infecting organisms’ susceptibility. In many countries free of contagious agalactia a slaughter policy is in place.

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

  Various wild small ruminants can be infected and act as carriers, but the disease caused by M. agalactiae is not considered a particular problem among endangered species. However, a fatal outbreak of pneumonia in endangered wild markhors in Tajikistan in 2010 was attributed to M. capricolum subsp. capricolum. Various wild small ruminants can be infected and act as carriers but not considered a particular problem among endangered species.

  GAPS :

  Wild ungulates are not specifically monitored for contagious agalactia in many countries, despite clinical disease occurring in ibex in the French and Italian Alps and in the Sierra Nevada region of Spain, with chamoix and wild markhors also affected.

  Plausible aetiological cofactors associated with the expression of observed clinical disease in wild ungulates requires investigation.

• Slaughter necessity according to EU rules or other regions

  In many countries free of the disease slaughter is used following identification of infected flock/herd or import consignment. Euthanasia is used for acute cases if death does not intervene already.

  GAPS :

  Contagious agalactia caused by M. agalactiae is notifiable to the OIE, but not when caused by other mycoplasmas, so slaughter would not necessarily follow in disease-free countries.

  Tertiary legislation relating to the omission of contagious agalactia in the new Animal Health Law Regulation EU 2018/1882 remains to be finalised.

• Geographical distribution and spread

• Current occurence/distribution

  The disease caused occurs in Europe (mainly confined to Mediterranean), East and Western Asia, South America, and North (and west) Africa. No recent cases have been reported in the United States of America and in Australia recovery of the organisms, but not clinical disease, has been reported.

  GAPS :

  True prevalence of contagious agalactia caused by M. agalactiae, (or by the other disease agents) in countries in which the disease is present as endemic/epizootic is not known; WAHIS reports outbreaks of disease without identifying the etiological agent, while some countries report only non-quantitative data. There are reports in the literature of cases in countries that are not included on WAHIS.

  Lack of diagnostic facilities, expertise and tools in many developing regions, means the disease is under-reported.

• Epizootic/endemic- if epidemic frequency of outbreaks

  Endemic in some parts of Southern Europe, parts of Eastern and Western Asia, America and North Africa.

  GAP :

  There remains an absence of disease monitoring networks in many countries.

• Seasonality

  Seasonal by association with young offspring.

  GAPS :

  Post-partum period and onset of lactation is time of greatest risk of appearance of disease.

  Nursing lambs and kids are highly susceptible.

  Winter/poor weather link to respiratory cases.

  Hot/dry season association with poor hygiene, nutrition and gathering of animals at waterholes.

• Speed of spatial spread during an outbreak

  Potential to be rapid. Likely to be dependent on animal welfare and the removal, or minimising, of risk factors in the environment.

  GAPS :

  Data detailing infection spread are generally still lacking.

  Spatial spread of clinical disease is observed, but detailed studies to evaluate spread of infection and monitor management / environmental risk factors, are required.

  Some infections may be missed if clinically inapparent and animals not removed.

  Impact of heat treated colostrum in reducing infection in off-spring from infected ewes or does needs further evaluation.

• Transboundary potential of the disease

  Environmental and aerosol transmission can occur along with carrier animals brought in, so there is a trans-boundary potential. OIE recommendations, for importation of sheep and goats, may leave countries that are normally free of disease vulnerable to importation of asymptomatic carriers, which are difficult to detect by available serological tests.

  GAP :

  Disease is spread, invariably, by animal movements.

  Potential threat may be associated with changes to regulatory categorisation of contagious agalactia following omission of the disease in the new Animal Health Law Regulation EU 2018/1882.

  Risks of transmission through infected germplasm need to be assessed.

  Development of more sensitive screening tests and/or use of screening tests for animals brought in.Design and development of improved surveillance strategies that are risk-based, would be appropriate, but likely to be expensive.

• Route of Transmission

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

  Horizontal transmission by animal (oral, respiratory/aerosol, mammary), environmental contact (bedding, milking parlour, feeding areas and implements). Vertical transmission is via mother to off-spring during pregnancy and through suckling. Use of infected rams/bucks, failure to separate young from infected mother, failure to implement hygienic practices generally, but specifically, to clean accommodation and milking equipment, are considered key risk factors for transmission.

  GAPS :

  Acceleration of spread of infection in the flock/ herd associated with milking (manual/automated).

  Length of long-term asymptomatic carriage (and associated shedding patterns) is not known.

  The importance of spread by normal human activity (vets, farm workers, vehicle movement) is unknown.

• Occasional mode of transmission

  Aerosol transmission is possible over short distances. M. agalactiae is also excreted in faeces and in urine which are not samples usually considered when testing for the organism.

  GAPS :

  The significance of aerosol transmission and pneumonia preceding bacteraemia and development of mastitis should be investigated.

  Transmission of infectious fluids from the eye through contact and/or by flies.

  Risks associated with genital transmission and infected germplasm.

  Extent of protection from infection offered to newborns by use of heat-treated colostrum from infected mothers.

  Reservoirs of M. agalactiae may persist in the farm environment with potential risk for naïve animals. There is a lack of knowledge of survival time and length of time required before restocking can safely take place, without risk of reinfection, when slaughter has been carried out.

• Conditions that favour spread

  Poor husbandry and hygiene, stress, improper maintenance of milking equipment. During dry season, poor nutrition may increase susceptibility and promote spread, as can mingling with potentially infected animals when feeding /drinking. Analysis in the Middle East also shows purchase of replacement animals and visits by veterinarians and other workers may also increase risk.

  GAPS :

  Lack of awareness of the disease and importance of hygienic measures.

  Improved knowledge relating to the impact of differing management strategies on long-term outcomes is required.

• Detection and Immune response to infection

• Mechanism of host response

  Following bacteraemia a serological response is noted, although in view of the prolonged shedding post infection this response is potentially inadequate, with the innate immune response apparently unable to control infection in experimentally infected animals. Asymptomatic carriers present challenges for detection through currently available antibody-based tests. The role of cell-mediated immunity has not been well established generally for mycoplasma infections. Recent M. agalactiae experimental infection studies on goats and sheep indicates that CD4+ and CD8+ memory interferon-γ+ T-cells proliferate at different rates during early stages of infection. Moreover, the ratios appear to change over time, with CD4+ peaking earlier, at 15 days post -infection (Castro-Alonso et al, 2010; La Manna et al, 2011). Although host neutrophils appear to respond to presence of M. agalactiae, and specifically its exposed lipoproteins, by forming neutrophil extracellular traps in mastitic mammary glands of infected sheep, enzymes excreted at the cell surface, such as the surface nuclease MAG_5040 (Cacciotto et al., 2016), appear capable of degrading the traps, thus having potential to overcome this first line of defence.

  GAPS :

  Further work on adaptive and innate immunity is required for M. agalactiae (and other agents of contagious agalactia). Data relating to host immune response to the other contagious agalactia causative agents is lacking and this area of work requires attention.

  For cell mediated immunity use of comparable approaches to improve understanding of the role of interferon γ cell proliferation and subsequent decline in the protective immune response in sheep infected with M. agalactiae would be beneficial. Moreover, understanding how this organism activates neutrophils and escapes from their antimicrobial activity, warrants further investigation. Protection offered by humoral response: although not believed to be protective, serum collected from recovered animals inhibits growth in vitro, in contrast with that from vaccinated comparators. Data from cell mediated immunity studies is unclear.

• Immunological basis of diagnosis

  Serological tests, for detecting the presence of antibodies are only widely available for M. agalactiae.

  GAPS:

  Serological tests are lacking for the other aetiological agents.Asymptomatic carriers of the herds tend to be serologically negative. Early stages of infection are also challenging to detect, as they invariably lag behind detection of the organism in milk.

  No specific host markers of infection have been reported. However, evidence is accumulating for a correlation between expression of microRNA molecules (miRNAs), circulating in the blood, and bacterial infection that may be exploited for future development of disease-specific biomarkers.

• Main means of prevention, detection and control

• Sanitary measures

  The infecting organisms can be introduced to a herd/flock by asymptomatic carriers. Sanitary conditions are important for minimising spread of disease. Disinfection can be effective against mycoplasmas. Reducing mammary transmission by disinfection of milking equipment, cleaning of teats, milking infected animals last and ensuring hand hygienic measures are practiced. Limited laboratory studies to date have mainly focused on clean cultures, with little done to determine the potentially inhibitory effect of organic matrices, with associated biofilms.

  GAP :

  The impact of organic matrices and biofilms on effectiveness of commonly used disinfectants, to reflect use in the farm environment and on milking equipment, should also be investigated.

  

• Mechanical and biological control

  In practical terms this is limited to preventing introduction of infected asymptomatic replacement stock into the flock or herd, adequate maintenance and disinfection of milking equipment, and the use of antibiotics and vaccination may be used.

  GAP :

  Insecticides may be an additional control method for biting insects and ticks. However, widespread use in agriculture is a risk for promoting resistance in vectors carrying zoonotic agents and would need to be carefully assessed.

• Diagnostic tools

  Definitive diagnosis is mainly by isolation of the organism. Traditional methods for identification including growth inhibition, metabolism inhibition, epi-immunofluorescence and peroxidase tests have largely been replaced by PCR-based techniques, although culture remains the gold standard for confirming index case of a disease outbreak.Indirect diagnosis with commercial ELISA tests exist only for M. agalactiae.

  GAPS :

  Serological screening by ELISA offers the most cost effective and sensitive method of preliminary diagnosis, as well as monitoring disease absence. However, these tests are unlikely to detect asymptomatic carriers and early stages of infection. Improvements needed in sensitivity to detect these animals. Experimental work developing host markers of infection for other mycoplasmas is in its infancy.

  Conventional culture based tests for identification of mycoplasmas are slow and laborious and are largely being replaced by molecular methods. Use of bulk-tank or pooled milk samples in countries for screening, although effectiveness of DNA extraction method against inhibitors and sensitivity of detection for use in low prevalence areas would need to be assured. Inter-laboratory evaluation of cut-off values, for direct detection of samples used for screening would be beneficial to optimise accuracy of interpretation.

  Lack of single definitive test to identify all four species. A multiplex PCR which can subdivide M. agalactiae from the other three agents of contagious agalactia is available commercially, but cost is likely to limit wide-scale use, particularly as a screening tool.

  

• Vaccines

  Live attenuated and inactivated vaccines are available. Commercially available vaccines comprise single strain M. agalactiae or as mixed M. agalactiae and M. mycoides subsp. capri / M. capricolum subsp. capricolum.

  High quality datasets regarding efficiency of vaccination protocols is lacking.

  GAPS :

  Live vaccines are not approved for use in Europe, despite good results in Turkey with an attenuated strain. Further independently validated studies are needed to determine efficacy of inactivated vaccines. Recent comparative studies (Agnone et al, 2013; Ozdemir et al, 2019) show the live vaccine to be most effective, despite a lack of detectable serological response. However, the animals may still excrete the infective strain in milk, with the risk of promoting spread of M. agalactiae from vaccinated flocks to naive unvaccinated animals. The combined vaccines, typically formalin inactivated, are used to control contagious agalactia in goats and appear to reduce spread of infection, but protection offered is otherwise limited.

  Independently conducted comparative trials of commercial vaccines are required to determine efficacy, while standardising requirements for evaluating efficacy would be beneficial.

  Improved vaccines with DIVA tests are needed.

  

• Therapeutics

  Lack of a cell wall restricts antimicrobial classes that are suitable for treatment of M. agalactiae and other mycoplasma infections. Moreover, the number of products licenced for small ruminants is relatively limited. Antibiotics are used; mainly tetracycline and macrolides, with florfenicol, tiamulin less commonly used in recent years. While treatment can resolve clinical signs, antibiotic use often fails to completely eliminate the causative mycoplasmas, The newer fluoroquinolones, the only class in use that is mycoplasmacidal, have had their application restricted in food-producing animals because of their use in human medicine.

  GAPS :

  Rarely completely eliminates causative mycoplasmas though can resolve clinical signs: field efficacy studies lacking.

  May encourage carrier state, but controlled studies are needed to determine this.

  Impact of improved herd management practices implemented in combination.

  Uncontrolled used may result in unnecessary antimicrobials entering the food chain and environment, in turn impacting susceptibility for other bacteria. Instead, giving vaccines to infected sheep may have an impact on the progression of the disease (Loria et al, 2018), an observation warranting further study.

  Some countries have reported reduced susceptibility to antimicrobial classes commonly used in treatment. The mechanisms conferring some, but not all, changes in susceptibility changes have been determined, with role for other potential mechanisms unexplored, for clinical isolates.

  

• Biosecurity measures effective as a preventive measure

  Important for preventing the introduction of M. agalactiae and other agents of contagious agalactia. Serological screening of flocks prior to purchase would greatly reduce risk, although identifying asymptomatic carriers, which may be difficult to detect serologically remains challenging. Introduced animals should be separated and monitored. This may need to comprise repeat microbiological testing of individuals. Mixed infections, comprising M. agalactiae and other agents of contagious agalactia are possible, particularly where both sheep and goats are reared (Al-Momani et al, 2006). General measures of herd management are important, not only to reduce contact with potentially infected animals and reduce levels of the infective agent in accommodation and the environment, but in reducing susceptibility to infection that may be predisposed by stress. Hygienic milking practices, with disinfection of milking equipment are also essential.

  GAPS :

  Effectiveness of detection of serological screening of flocks prior to purchase in reducing risk, particularly associated with asymptomatic carriers.

  The prevalence of mixed infections is unknown, but should be considered.

  Improved knowledge of survival of the organism in the environment and effective disinfection in organic matrices.

  It would be beneficial to determine experimentally whether the reduction in load of M. agalactiae offered by heat-treated colostrum is effective in preventing transmission.

• Border/trade/movement control sufficient for control

  The OIE Animal Health code recommends animals should have an international veterinary certificate certifying the animal showed no clinical signs on day of shipment; that the animal was kept since birth or for 6 months prior to shipment in an establishment where no case was officially reported during the period, and that the animal was kept in a quarantine station for 21 days prior to shipment. Post-importation /exportation serological testing is required for disease free countries.

  GAPS :

  Serological detection before shipment should be performed, although improvement of tests for detection of asymptomatic carriers would also be beneficial.

  A difficult sample to reproducibly collect, the significance of ear carriage in sheep and goats should be defined, as should carriage in semen and other germplasm.

• Prevention tools

  Awareness of the disease, general hygiene and herd management, including removal and replacement of bedding, maintenance of milking equipment, regular inspection of animals for clinical signs are important in preventing disease introduction and for containment within an infected herd/flock.

  GAPS :

  Vaccination of replacement or new stock before introduction to flock/herd.

  Systematic detection of the mycoplasmas should be performed at the flock/herd level before animal introduction into a free area/flock.

  In enzootic areas, extension of systematic infectious status definition for flocks is limited by the cost of direct/indirect detection.

• Surveillance

  Contagious agalactia is an OIE notifiable disease.

  GAPS :

  Notification to OIE appears to be overlooked by some countries, thwarting assessment of true distribution and prevalence.

  Post-importation /exportation serological testing required and used as part of existing international collaboration for control across borders. The impact of EU’s new proposals 2016/429 may downgrade the disease to national or even local importance. Such a development could affect cross border control and make this a less attractive area for research funding.

  The true incidence of contagious agalactia caused by M. agalactiae or other causative agents, individually, is not really known.

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

  Although contagious agalactia has been eradicated from some countries it is difficult to control once it has established and becomes widespread; asymptomatic carriers help maintain transmission to naïve animals. For some countries (e.g. Australia) contagious agalactia causative organisms seem to be carried without clinical disease being evident in the small ruminant population.

  GAPS :

  Eradication (without vaccination) was successfully achieved in French Alps (goats). In the French Pyrenees prevalence has been reduced to 5%, difficulties have included the density of sheep population and difficulties associated with detection of asymptomatic carriers which help to maintain the disease.

  Reasons for failures include difficulty of applying sanitary measures, absence of efficient vaccine and costs of lab diagnosis.

  Serological screening is an essential tool. However, difficulties detecting asymptomatic carriers and infections caused by antigenically divergent strains pose a threat to surveillance screening programmes.

  Host disease resistance.

• Costs of above measures

  Expensive: The costs of treatment, culling (and stock replacement), control, eradication, or prevention of the disease are significant. Of note: there is a current trend in increasing goat and sheep breeding in different countries worldwide. The cost of the measure should be more accurately defined to be taken into account.

  GAPS :

  Cost of disease to Greece was estimated at 25 million euros in 1995. Costs of enforcing regulations in Italy are high requiring the slaughter of stock when disease confirmed, a recent estimate of the cost to the farmer of controlling a disease outbreak in the flock of 1000 sheep in Italy, including killing of clinically affected animals, impact on dairy production, expenditure on disease control (vaccination), veterinary costs and restocking was estimated at over 100,000 Euros (Loria and Nicholas, 2018). A survey including all similar cost factors in other countries, including traditional and more modern production systems would provide useful evidence.

  PCR is necessary, but expensive to monitor at individual animal level; on farm isothermal tests are under development. Bulk tank milk an alternative sample, but sensitivity with tests in use and variation in outcomes associated with DNA extraction efficiency, is of some concern. In comparison, the costs of stamping out through culling is both genetically and financially expensive.

  Early recognition of disease for earliest treatment (or culling) and segregation from healthy animals is required.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  Yes. The OIE list 859 reports notified of contagious agalactia in the 18 month period from January 2018, the vast majority being in Iran (60.6%), followed by Italy (14.3%) and Mongolia (9.9%).

  GAPS :

  Not all countries report data to the OIE, despite description of cases of the disease in the literature, the OIE WAHIS data does not differentiate between agents of contagious agalactia and the outbreak data submitted by some countries is not quantitative.

  Under reporting estimated at 90% in S Italy because of strict regulations which seriously affect farmers’ incomes.

• WOAH disease card available

  No

• WOAH Terrestrial Animal Health Code

  Link to OIE

• WOAH Terrestrial Manual

  Link to OIE

• Socio-economic impact

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

  Not applicable as not proven as zoonotic agent. An increased use of antibiotics to treat the disease could lead to increased resistance among human pathogens.

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

  N/A.

• Direct impact (a) on production

  Loss of milk production, and associated products, as a result of clinical and subclinical mastitis, increased mortality with abortion also possible. Mortality is normally higher in goats, and following infection with other CA agents, and thus has a serious impact on farms in the endemic areas particularly where traditional production methods are practiced.

  GAP :

  Recording of milk yield needed to assess economic cost at a flock/herd level.

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

  Significant: compensation schemes may be available in some countries where contagious agalactia is absent, should a disease incursion occur. Where disease is endemic results in significant cost to the farmer.

• Indirect impact

  In the endemic areas it is not considered that a significant fall in tourism, security in food supply or constraints on livestock production would be significant.

• Trade implications

• Impact on international trade/exports from the EU

  For those EU countries where the disease is present the OIE Animal Health Code regarding transporting animals would need to be addressed. Establishment of disease in a previously free country would have substantial impact on the ability to trade in live animals and germplasm.

  GAPS :

  Omission from the Animal Health Law due to be implemented in April 2021, as originally published in 2019, raises further concerns regarding to the importance placed on the disease, with consequences for existing international border control cooperation.

  Additional pre-import/post-import testing for disease free countries and development of more sensitive tests to detect healthy carrier animals should be considered.

• Impact on EU intra-community trade

  For those EU countries where the disease is present the OIE Animal Health Code regarding transporting animals would need to be addressed.

• Impact on national trade

  For those EU countries where the disease is present the OIE Animal Health Code regarding transporting animals would need to be addressed.

  There are increasingly pressing demands on breeders to be able to establish a “herd status” with respect to mycoplasmosis to secure exchanges in between herds.

• Main perceived obstacles for effective prevention and control

  Awareness of this notifiable disease, and associated risks, to the welfare of the domestic small ruminant population is still inadequate, with a lack of international agreement on the way forward in tackling the disease as farming practices intensify in developing regions. Once the disease becomes established in a flock or herd, it can be difficult to eradicate, with the most commonly used antimicrobials acting in a mycoplasmastatic way and may not eliminate the organism. Sub-clinically infected animals and carriers among those infected bearing in mind the extended period of shedding (potentially up to 8 years).

  GAPS :

  The significance of ear and semen carriage, and of pneumonia as sources of spread needs to be addressed.

  Transmission via biting insects, ticks and mites should be investigated.

• Main perceived facilitators for effective prevention and control

  Vaccination is considered appropriate. However, the efficacy of current vaccines is a matter of concern. Live attenuated vaccines are considered more effective than inactivated vaccines but live vaccines are not permitted in some of the affected countries.

  Research has focused on identifying immunogenic proteins that can be utilized in subunit vaccines and/ or improved serological tests. Recent advances including comparative genomics with proteome analysis and epitope mapping are making it much easier to identify and locate immunogenic proteins while transposon mutagenesis has facilitated improvements to our understanding of the role of the surface exposed vpma lipoproteins in adhesion, invasion and immune evasion. The recent advancements in synthetically engineered genomes developed for the Mycoplasma mycoides cluster, which includes two causative agents of the disease, is facilitating construction of genetically attenuated strains.

• Links to climate

  Seasonal cycle linked to climate

  This has not been generally recognised, but bearing in mind the geographical distribution, this may be a possibility due to stress caused by changes in husbandry, nutrition, particularly among adult flocks/herds in dry season. Greater risk of aerosol transmission may promote spread by inhaled aerosol, particularly where animals mingle with potentially infected animals at waterholes.

• Distribution of disease or vector linked to climate

  Not generally recognised, but bearing in mind the geographical distribution and potential for biting insect, or tick, vectors this may be a possibility.

• Outbreaks linked to extreme weather

  Not generally recognised, but considering geographical distribution and climatic effects, the gathering of animals at watering holes in countries prone to drought could increase likelihood of contact with infected animals. Moreover, associated stresses may enhance vulnerability to infection. Flooding could encourage environmental survival.

  GAP :

  Links to changes in climatic conditions, or sudden weather events, with occurrences of disease.

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

  This has not been generally recognised, but bearing in mind the geographical distribution and recent evidence to suggest climatic changes this is of potential concern. Expanding grazing range and gathering of animals at reducing numbers of watering holes associated with climatic change and drought would increase likelihood of contact with infected animals and associated stresses would enhance vulnerability to infection.

Risk

• Contagious agalactia is a highly infectious disease of sheep and goats which is one of 117 diseases listed by the OIE. The current geographical location of the disease, caused by M. agalactiae, with three other agents important in goats, indicates prevalence in sub-tropical regions. Disease is invariably spread by movement of animals. OIE recommendations, for importation of sheep and goats, may leave countries that are normally free of disease vulnerable to importation of asymptomatic carriers, which are difficult to detect by available serological tests. The current geographical location of the disease suggests it is prevalent in sub-tropical regions; climatic changes may lead to diversification by farmers in small ruminant dairy farming and in turn may enhance potential for this disease to spread to currently clear areas. The use of antibiotics to treat the disease also may, not just encourage asymptomatic carriage with risk of intermittent shedding, but may lead to increased resistance among human pathogens and is an indirect risk.

  GAP :

  See Section “New developments for vaccines - Requirements for vaccines development/ main characteristics for improved vaccines”

Main critical gaps

• • Knowledge of the true incidence, contribution of the different causative agents in goats and the economic cost of CA disease.
  • Still lack of awareness of the disease. Good practice guidelines for disease prevention and outbreak management are needed.
  • Paucity of rapid, affordable, screening tests with improved sensitivity for asymptomatic carrier animal.
  • Lack of affordable standardised tests, with regular quality controls/ring trials.
  • Updated regulation in countries where severity of restrictions constrains outbreak reporting.
  • Improved knowledge on the factors associated with reactivation of Mycoplasma, pathogenicity mechanisms, including the contribution of the host immune response to lesion development, to unusual clinical signs and to absence of disease in some young animals.
  • Further work on the transmission mechanisms: the role of pneumonia, significance of germplasm and aerosol transmission, role of biting insects and ticks, excreta and the environment.
  • Contribution of horizontal transfer of genetic material demonstrated in M. agalactiae to observed strain diversity, global disease epidemiology and reduced susceptibility to therapeutic antimicrobials.
  • Lack of globally accepted, effective and independently trialled vaccines. A marker vaccine together with a suitable diagnostic means of distinguishing between vaccinated and infected animals is needed. Newly developed engineering tools for development of synthetically constructed attenuated strains and trialled with the M. mycoides subsp. capri genome show early promise for future mycoplasma vaccinology.
  • Standardised in vitro testing of antibiotics and monitoring of antibiotic effectiveness/resistance. Antibiotics with increased efficacy/distribution that could prevent the continued excretion of mycoplasma. Screening and in vivo assessment, using standardised evaluation procedures, of novel chemicals and plant extracts.

Conclusion

• Despite being known for over 200 years and the economic and welfare burden associated with contagious agalactia, there is still a lack of awareness in many parts of the world. Mycoplasma agalactiae remains the most important aetiological agent particularly in sheep, although in goats other causative agents appear to have increased in importance within some European countries, or regions. Rapid real-time PCR tests are increasingly used for confirmation and an international comparison of performance for different clinical sample types would be appropriate. A commercial PCR that differentiates M. agalactiae from other causative agents is available, but is currently expensive and doesn't differentiate between the disease agents belonging to the ‘M. mycoides cluster’. Isothermal tests aimed at on-farm testing are under development, but yet to become commercially available. Slaughter of affected flocks/herds, chemotherapy and/or vaccination are used to control the disease, but methods of control are regulated at country level, lacking an internationally coordinated approach. Limited effectiveness of antibiotics, which can alleviate clinical signs, but may fail to clear the organism, with sub-clinically infected animal shedding viable organisms for months and even years. Live attenuated vaccines are not permitted in Europe and are unsuitable for lactating animals, while the effectiveness of inactivated commercial and autogenous vaccines is hotly debated. Suitable vaccines would need global acceptance. Commercial ELISA tests are available for M. agalactiae, but not disease caused by other causative agents. Difficulties remain with detection of clinically healthy carriers, in which the organism appears to evade the immune system. Interaction, and effect on disease severity, of multiple infections with the four causative mycoplasma organisms need investigating. Genomic exploration to investigate recent evolution of strains is also required; while MLST data is available whole genome sequenced based comparative analyses are lacking for all aetiological agents of CA and consequently there is poor data comparability worldwide.

  GAPS :

  The following factors summarise the problems:

  Greater global awareness, availability of cost-effective in-field tests will improve knowledge of disease prevalence associated with this economically important disease is still required.

  Widely accepted effective DIVA vaccines, evaluated using harmonised regulatory procedures are required.

  Improved ability to detect latent infections and asymptomatic carrier animals which present a major risk for naïve herds/flocks, is required.

  Once established in a flock or herd it can be difficult to eliminate; despite generally low MIC values to commonly used antimicrobials, treatment may not eliminate the organism and encourage carrier state.

  Harmonisation of restrictions applied at country level following identification of outbreaks in countries where the disease is endemic will be welcomed.

Sources of information

• Expert group composition

  Anne Ridley, APHA, UK – [Leader]

  Florence Tardy, ANSES; Vet Agro Sup, France

  Guido Loria, IZS, Sicily, Italy

  Roger Ayling, APHA, UK

  Robin Nicholas, UK.

  The group consulted with:

  Marc Marenda, University of Melbourne, Australia

  Mercè Domènech, HIPRA,Spain

  Anna Greatrex, IDVET, France.

• Reviewed by

  Project Management Board.

• Date of submission by expert group

  10/09/2020

• References

  Updates to current knowledge of CA and its different etiological agents was recently reviewed in

  Jaÿ, M. and F. Tardy (2019). Contagious agalactia in sheep and goats: current perspectives. https://doi.org/10.2147/VMRR.S201847

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