Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  Kits for serological diagnosis are available that can only be interpreted at herd level: complement fixation test (CFT) and competition ELISA (cELISA).

  • CFT is produced by the WOAH reference laboratory for CBPP at CIRAD, Montpellier (France) and by a private company. Mycoplasma mycoides subsp. mycoides (Mmm) antigen and control sera for CFT are also available from the WOAH reference laboratories for CBPP in Oeiras (Portugal), Teramo (Italy) and Gaborone (Botswana).
  • cELISA based on reagents developed at CIRAD (Le Goff and Thiaucourt, 1998) was previously commercialised but production stopped. A new cELISA based on the same technology is under development for reintroduction to the market.
  • Contagious Bovine Pleuropneumonia Antibody (Anti-CBPP) ELISA Kit. This indirect ELISA test kit is for research only. No field validation data is available. It is therefore not recommended for diagnostic purposes.
  • Latex Agglutination Test: For rapid screening at the pen-side, allowing diagnosis of acute cases in remote areas; limited specificity. Its production ceased due to high cost.

  Commercial culture media that support the growth of Mmm are available:

  • Mycoplasma medium broth: sterile liquid base to be stored at room temperature (RT) and frozen supplement at -20°C.
  • Mycoplasma agar/broth base and Supplement G: dried base, to be stored at RT and autoclaved upon reconstitution, and lyophilised supplement to be stored at +4°C.
  • CBPP diagnostic medium: sterile liquid base to be stored at RT and frozen supplement at -20°C.
  • Alternative Mycoplasma/PPLO bases are also available but their corresponding lyophilised supplements are extremely expensive. Alternative supplements may be used but this would require validation.

  There are some commercial real-time PCR kits available, though the target sequences are not known.

  GAPS :

  In the countries where CBPP is endemic there is a decrease in the use of diagnostic tests due to reduced veterinary and laboratory capacity as a result of logistical, financial and political constraints. This is particularly alarming in countries suffering from civil war and other armed conflicts, as is the case of the central Sahel and the Horn of Africa, where the security situation has deteriorated dramatically due to the emergence of terrorism.

  In the last couple of years there has been a severe shortage of CBPP cELISA kits and production has definitely ceased in 2023. There is an urgent need for alternative, highly specific ELISA kits for use in international trade as well as for field surveillance and prevalence studies.

  No commercial pen-side tests are available for rapid CBPP diagnosis in the field.

  The offer of commercial culture media for Mmm isolation and antimicrobial susceptibility testing is very limited and their availability is low (shortages and long delays to supply are common). Mycoplasma media are expensive, particularly lyophilised supplements. Furthermore, supplement storage requires strict conservation of the cold chain either frozen (liquid form) or refrigerated (lyophilised formulations).

  Neither data on performance (specificity and sensitivity) nor on field or inter-laboratory validation are available for the commercial PCR kits.

• Diagnostic kits validated by International, European or National Standards

  Diagnostic kits have been validated by CIRAD and are listed in the WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 3.4.8 (WOAH, 2021): CFT (Amanfu et al. 1998) and cELISA (Le Goff and Thiaucourt 1998, Amanfu et al, 1998).

  No diagnostic kits have been validated by European standards but the CBPP cELISA (IDEXX) has been validated by the French Committee for Accreditation (COFRAC) in 2009. Unfortunately, this kit is no longer produced.

  GAPS :

  No diagnostic kits have been validated by European standards and there is currently no European reference laboratory for CBPP.

  Both direct molecular assays and serological kits must be validated by international standards.

  International inter-laboratory assays must be carried out to compare the performance of direct detection and serological diagnostic assays, notably to fully validate an alternative CBPP cELISA kit.

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

  Routine diagnostic methods are described in the WOAH terrestrial manual (WOAH, 2021):

  Serological methods:

  (a) CFT (Campbell and Turner 1953): suitable for determining freedom from disease and a prescribed test for prevalence studies and international trade; with a sensitivity of 63.8% and a specificity of 98% (Bellini et al., 1998).

  (b) CBPP cELISA: prescribed for the same purposes as CFT, with similar sensitivity but higher specificity: 99.9% (Le Goff and Thiaucourt, 1998; Amanfu et al., 1998).

  (c) Immunoblotting test (Gonçalves et al. 1998): Very high specificity; most useful as a confirmatory test but not fit for mass screening. Difficult to standardise, as many factors can influence the final banding pattern (Gaurivaud & Poumarat, 2012). This test has insufficient robustness and reproducibility and requires highly skilled personnel.

  (d) Slide agglutination and latex agglutination test (LAT): for rapid confirmation in the field, using either coloured antigen or sensitised latex beads, which yield agglutination in the presence of positive sera containing IgMs.

  Direct detection and identification:

  (a) In vitro culture: isolation of the CBPP agent remains the prescribed test for disease confirmation. The composition of a culture medium for growth of Mmm is provided in the WOAH manual. In broth, growth is visible by development of slight turbidity and forms silky swirls when shaken. On solid media small colonies are seen, presenting a classical ‘fried-egg’ appearance. Mmm does not produce film and spots.

  (b) Biochemical and immunological identification tests: Special media for biochemical tests are described in the manual, as well as the biochemical characteristics of Mmm: sensitivity to digitonin, reduction of tetrazolium salts, fermentation of glucose, absence of arginine hydrolysis, and absence or weak phosphatase and proteolytic activities. However, biochemical characterisation is not sufficient to allow precise mycoplasma identification and subsequent immunological tests are needed. The main methods listed in the manual are: the growth inhibition test, the indirect fluorescent antibody test, the agar gel immunodiffusion test, and the dot immunobinding on membrane filter test. At present, few laboratories use biochemical and immunochemical tests for identification of Mmm, since molecular methods are more specific, sensitive, rapid and easy to perform and standardise (WOAH, 2021).

  (c) Molecular detection: the conventional PCR method based on amplification of the CAP-21 genomic fragment, followed by restriction enzyme analysis of the PCR product (Bashiruddin et al. 1994) is described in the manual, while other molecular detection methods are listed: Conventional PCR methods include a specific PCR test for direct detection of Mmm (Dedieu et al., 1994) and a nested PCR test based on the lppA gene (Miserez et al., 1997), while Lorenzon et al. (2008) is a SYBR Green-based real-time PCR. Three additional real-time PCR methods have been published, though they are not listed in the manual: Two TaqMan-based: Gorton et al. (2005), and Schnee et al. (2011); and another SYBR Green-based (Fitzmaurice et al., 2008).

  GAPS :

  Potential specificity problems have been identified with most of the molecular diagnostic assays described here since the publication of the complete genome sequence of a Mycoplasma mycoides subsp. capri (Mmc) strain isolated from a goat in India (strain Ker TCR LT, Accession: NZ_CP068548.1), which presents typical Mmm sequences in most of the molecular targets published to date. The only assays that remain specific in regards to this Mmc strain (based on the presence of a number of SNPs in their target sequence allowing a possible differentiation) are (i) the CAP-21 PCR-based test of Bashiruddin et al., (ii) the nested PCR test of Miserez et al., and (iii) one of the three TaqMan real-time PCR primer-probe combinations of Gorton et al. targeting a hypothetical lipoprotein. Having said that, neither the prevalence of such cross-reacting Mmc strains in Asia and other parts of the world nor their level of circulation in cattle are known.

  Limitations of “Miserez” and “Bashiruddin” PCR-based assays: The assay described by Miserez et al. is a nested PCR and is thus subject to contamination problems when used for routine diagnosis. The assay described by Bashiruddin et al. is not a direct PCR test but requires restriction enzyme analysis of PCR products for specific diagnosis (for differentiation of Mmm and Mmc strains). This does increase labour and time, and can lead to interpretation problems as well as increasing the risk of contamination. As a consequence, the real-time PCR assays should be preferred.

• Commercial potential for diagnostic kits in Europe

  The market is limited, especially as CBPP is only present in sub-Saharan African countries, where affordability is limited. In these countries the conditions for delivery and conservation may be an issue, so the thermostability and robustness of diagnostic assays is of great importance.

  GAPS :

  Research is needed to develop and validate highly effective tests, particularly pen-side tests.

  Market research is necessary to assess demand and affordability of new diagnostics.

• DIVA tests required and/or available

  No DIVA test exists since no DIVA vaccines are available.

  GAPS :

  Research is needed to develop and validate DIVA tests, in line with DIVA vaccine developments (market studies may be needed to assess this).

• Vaccines availability

• Commercial vaccines availability (globally)

  Only live attenuated vaccines are available for CBPP. Although other Mmm strains have been used previously (e.g., KH3J in Africa, Ben-1 in China and V5 in Australia) only the T1 strains are currently in use and commercially available (i.e., T1/44 and its streptomycin-resistant derivative T1sr).

  Veterinary vaccine batches to be used in Africa are controlled by AU-PANVAC (Ethiopia) but to date this requirement is not mandatory for vaccine batch release and commercialization. Below producers have had vaccines certified by AU-PANVAC between 2014 and 2022:

  https://aupanvac.org/brizy-4163/certified-vaccines-batchs/

  Strain T1/44 is produced by NVI (Ethiopia), JOVAC (Jordan), Hester and TVLA (Tanzania), KEVEVAPI (Kenya), BVI (Botswana), MCI (Morocco), LANAVET (Cameroon), LABOCEL (Niger), LCV (Mali), ISRA-LNERV (Senegal), NVRI (Nigeria), Laprovet (France https://lobs.fr/produits/peribovac/)

  Strain T1sr is produced by NVI (Ethiopia), JOVAC (Jordan), LANAVET (Cameroon), and LCV (Mali).

  Quality control methods (QC) used by vaccine producers and AU-PANVAC: Titration, identity, purity (freedom from contamination with fungal, bacterial and viral agents), and safety (innocuity) in target species are conducted by vaccine producers.

  Safety in lab animals, vacuum and residual moisture check (in the lyophilised vials), identity by T1-specific PCR

  (Lorenzon et al., 2000), sterility and titration are assessed by AU-PANVAC.

  In many countries vaccination campaigns are organised by the public veterinary services (generally once a year) and farmer compliance is encouraged by offering antiparasitic treatments, and/or reduced vaccination prizes (in some cases totally free of charge). However, CBPP vaccines are not available to farmers (not even via private vets).

  Whether a combination of private farm-level management with government run campaigns would improve the control of the disease needs to be explored. Few studies on optimal policy on vaccine implementation strategies in CBPP control have been published (Onono et al., 2017; Suleiman et al., 2018).

  GAPS :

  The number of vaccine doses available is not always sufficient to satisfy the demand and allow massive vaccination campaigns for the protection of the entire cattle population in affected areas.

  Availability of vaccines in sub-Saharan Africa varies considerably depending on whether CBPP control is regarded as a private or a public good and depending on the economic situation of the country.

  As previously stated regarding access to diagnostics, logistical, financial and political constraints can greatly limit access to vaccines and vaccination campaigns.

  Furthermore, vaccines are not available directly to the farmers outside the frame of campaigns organised by the veterinary services. Involvement of private vets in delivery of CBPP vaccines to farmers should be considered.

  Independent QC on vaccine batches commercialised in Africa is not performed systematically and the number of vaccine batches undergoing QC by AU-PANVAC is very limited.

  Current QC procedures applied are limited to innocuity, purity and titres, since there is a minimum titre associated with protection. However, they do neither assess potency nor the level of genetic drift of T1 strains used for new vaccine batches.

  In the absence of small animal models, in vitro models, and/or biomarkers of vaccine efficacy that could serve as correlates of protection, assessing the potency of vaccine batches would actually require CBPP challenge experiments in cattle, which are not performed in practice.

• Marker vaccines available worldwide

  No marker vaccines are currently available but subunit and inactivated prototypes are under development:

  -Subunit vaccine (VIDO, ILRI, KALRO) (Nkando et al. 2016)

  -Negatively marked inactivated Mmm strain in oil adjuvant (Vacnada project, unpublished data).

  Recently developed Cas9 genome editing technologies may be applied to current T1 live vaccines to generate a negatively marked vaccine.

  GAPS :

  DIVA vaccines and companion tests are required (may need to start by performing market studies). DIVA vaccines are needed in countries such as Zambia, which have put eradication programmes in place including vaccination.

• Effectiveness of vaccines / Main shortcomings of current vaccines

  The protection rate of T1/44 and T1sr vaccines assessed in two independent vaccine trials varied between 33 and 67% (Thiaucourt et al., 2000). Animals were challenged against local pathogenic strains, 3 months after single vaccination. Further experiments demonstrated that T1/44 confers longer immunity than T1sr after a single dose, while a booster vaccination received 12 months after initial immunisation increased the efficacy of both vaccines (> 80%; Wesonga et al., 2000). In order to be effective vaccines must contain at least 10⁷ CFU/dose of live Mmm (WOAH, 2021).

  Both vaccines confer low efficacy at primo-vaccination and short-term immunity, requiring re-vaccinations every 6 and 12 months respectively for T1sr and T1/44. T1/44 has residual virulence, which results in occasional cases of severe inflammation at the site of injection called Willems’ reactions. These local reactions can evolve into systemic disease and may even result in death of the animals in the absence of antibiotic treatments, which can discourage vaccine acceptance by farmers (Teshale et al., 2005). Willems’ reactions are observed mainly upon first vaccination and with low occurrence after re-vaccination (Muuka et al., 2014). No side effects have been reported for T1sr.

  Both T1/44 and T1sr are currently produced as freeze-dried live vaccines with cold chain requirements until delivery. After reconstitution, delivery time is restricted to 30 min - 2 hrs. In field conditions and with hot temperatures, viability rapidly drops below the minimum recommended titre affecting vaccine efficacy.

  The quality of CBPP vaccines is known to differ among producers / batches, which may result in variable vaccine efficacy. Due to the potential side effects and cold chain requirements, vaccine delivery should be restricted to fully trained veterinary personnel.

  GAPS :

  Vaccine safety: Side effects associated with the T1/44 strain are perceived as an important issue. For example, T1/44 is not accepted in some countries due to reported adverse reactions and T1sr is used instead, although the frequency of vaccination campaigns is normally once per year. Vaccine delivery can exacerbate side effects, since applying inappropriate administration routes (intradermal or intramuscular instead of subcutaneous) or using inappropriate needle gauge, etc. can result in increased adverse reactions and, thus, reduced acceptability by farmers.

  Vaccine efficacy: The true efficacy of T1 vaccines in the field is not well known. Control programs based on vaccination alone are not sufficient for CBPP eradication in the absence of additional countermeasures such as animal movement control. For ex.: Northern Namibia and Western Zambia, where CBPP mass vaccination campaigns are delivered annually, are still struggling to control the disease, with many outbreaks declared in 2021 and 2022.

  Vaccine delivery: Fully trained personnel is needed to manage vaccination, since administration problems such as use of wrong diluents and vial sizes, inappropriate number of doses per vial, etc. can result in reduced viability and, thus, compromise vaccine efficacy.

  Thermostability: Cold chain requirements over storage and distribution and short time viability after reconstitution of the current vaccines limit operational activities for vaccine delivery.

  Duration of immunity: immunity is short-lived, which results in need of revaccinations (at least once a year for T1/44) throughout the life of the animal.

  Shelf life: No data are available regarding vaccine stability, notably remaining titres at the end of the shelf life and at the point of delivery. Long conservation (up to 2 years) requires freezing and shelf life is much reduced upon conservation at ~4°C.

• Commercial potential for vaccines in Europe

  The potential uptake of effective and safe vaccines by affected countries is very high due to extensive presence of the disease, causing high losses in sub-Saharan Africa

  The commercial potential of any new vaccine will be heavily dependent on its cost, but few studies have looked at willingness to pay and/or cost/benefit ratios (Tambi et al., 2006; Kairu-Wanyoike et al., 2014; Onono et al., 2014).

  GAPS :

  Market studies on improved formulations as well as new generation vaccines (subunit vaccines and live, genetically modified strains) should include acceptability of vaccine products (including GMOs), as well as willingness to pay (at farmer and governmental levels) and cost/benefit ratios.

• Regulatory and/or policy challenges to approval

  Registration of vaccines in Africa is currently fragmented, with many countries having their own specific policies. Restrictions for approval of novel GMO and recombinant vaccines will be particularly challenging.

  CBPP vaccine potency assessment is hampered by the lack of a reproducible in vivo challenge model. Efficacy evaluation of the current T1/44 vaccine varies widely between trials, partly due to the different strains and infection models used for challenge (further discussed in section 5.1).

  GAPS :

  Current regulations for vaccine registration should be adapted to include novel products such as GMO and subunit vaccines, since this is likely to limit access to the market.

  A reproducible in vivo challenge model is needed to evaluate the potency of current vaccine batches, as well as any new CBPP vaccines. Furthermore, a reliable batch consistency QC approach including in vitro efficacy of new vaccine batches should be developed and validated to minimise the use of live animal trials.

• Commercial feasibility (e.g manufacturing)

  Manufacturing of current CBPP live vaccines is not very challenging. Vaccines are produced upon demand (contract dependent). They are mainly distributed to governmental institutions in charge of organising vaccination campaigns.

  Yet some CBPP vaccines are produced in facilities not meeting international standards and using outdated processes for production and QC. They thus suffer from

  many shortfalls, including sterility issues, reduced shelf life and insufficient QC.

  GAPS :

  The production and QC of improved vaccines under the Good Manufacturing Practice (GMP) process by manufacturers needs to be promoted and adapted to new technologies.

• Opportunity for barrier protection

  In CBPP-free countries and zones of Southern Africa vaccination is prohibited and slaughtering policies are applied for disease control. However, in case of re-introduction of CBPP in certain regions, infected herds may be slaughtered, while herds in the surrounding regions are vaccinated in order to prevent further spread. Following the introduction/reintroduction of CBPP in different regions of the country, Zambia put in place an eradication strategy based on zoning, which involves mass vaccination in designated areas, including protection zones.

• Pharmaceutical availability

• Current therapy (curative and preventive)

  Currently, the use of antibiotics to treat CBPP is officially not permitted or at least strongly discouraged in most CBPP endemic countries, and this approach has been supported by WOAH and FAO for many years. The two main reasons for this are (i) the risk of development of antimicrobial resistance (AMR), particularly since these treatments are often applied under suboptimal conditions, and (ii) the notion that treatment of sick animals may favour the development of silent carriers by inducing the formation of encapsulated chronic lung lesions (known as sequestra) containing live Mmm, which may reactivate resulting in shedding and subsequent spread. Data to support the latter does not yet exist. Sequestra are often formed during the chronic stages of disease, but their role in reactivation and transmission of Mmm to other animals has not been demonstrated. Furthermore, formation of sequestra was not found to be more frequent in treated vs. untreated animals (see below). For what concerns the emergence of AMR, although no resistant Mmm strains have been reported to date, the increasing emergence of resistant Mycoplasma bovis strains is creating great concern (see section 20).

  Mycoplasmas are intrinsically resistant to many antibiotics (e.g., all that target the cell wall) and rapidly become resistant to aminoglycosides, which should therefore be avoided (Thiaucourt, 2018). The main antibiotic classes that have been shown to be effective against Mmm are tetracyclines, macrolides, lincosamides and fluoroquinolones.

  Very little in vitro data is available regarding Mmm sensitivity to different antimicrobials (Ayling et al., 2000; Ayling et al., 2005; Mitchell et al., 2012) and there are no approved standardised protocols and controls available for the determination of minimum inhibitory concentration (MIC) values of Mmm to currently used antimicrobials.

  Published in vivo trial data show that long acting oxytetracycline had an effect on reducing both clinical disease and the number of sequestra formed, although not entirely blocking the development of sequestra. It was also possible to isolate live Mmm in some sequestra, but not all (Niang et al., 2006; Niang et al., 2010, Yaya et al., 2004; Muuka et al., 2019). Two of the four studies testing the effect of oxytetracycline looked at the possibility of reducing transmission to in contact animals (Niang et al., 2004, Muuka et al., 2019). No transmission was observed, although in these trials either a control group was lacking (Niang et al., 2004) or very few animals (5/group) were included in the in-contact animal groups, resulting in very little transmission (1/5 and 2/5) observed in the negative control groups (Muuka et al., 2019).

  Huebschle et al., 2006 tested the effect of danofloxacin on CBPP infected animals in Namibia. No reduction in clinical disease was observed. Both the development of lesions and transmission to in-contact animals were reduced but not abolished, and it was still possible to isolate live Mmm from lesions. Whether the lesions were sequestra or other was not described.

  Finally, Muuka et al., 2019 tested the effect of two macrolides: tulathromycin and gamithromycin. Again, clinical disease and development of lesions were reduced, although not completely abolished. Isolation of live Mmm from lesions was possible, but greatly reduced compared to the control groups. No transmission was observed, but since transmission was low in the small control groups (as described above), this should be interpreted with caution.

  Although it is well known that cattle owners frequently resort to antibiotics when their animals get sick, often using oxytetracycline or tylosin (Kairu-Wanyoike et al., 2014, Mariner et al., 2006, Msami et al., 2001), publications describing their use in the field, specifically for CBPP, are rare. No benefit in the control of CBPP spread as a result of measures applied by farmers (including antibiotic treatment of sick animals) was evidenced in a field study conducted in Ethiopia. However, it was concluded that this might be related to reduced statistical power and low quality of both the medications and the health-care delivery system (Lesnoff et al., 2004). Kenyan farmers reported that treatment with tetracyclines often leads to relapse of the disease (Kairu-Wanyoike, et al., 2014). More recently, among various antibiotic treatments used in a field outbreak in Ethiopia, only tylosin prevented disease relapses (Almaw et al., 2016), while danofloxacin treatment in Namibia resulted in an immediate drop in cases, with no further disease reported during a two-year period (Nicholas et al., 2012).

  In addition to antimicrobials, clinical studies attempted to modulate the immune response of cattle exposed to CBPP infection by using Cyclosporin A (CsA), an immunosuppressive drug that is used to inhibit T cell activation (Scacchia et al., 2007). CsA did not prevent disease but clinical, humoral and pathological changes were delayed in treated animals.

  No therapeuticals have been validated for the preventive treatment of CBPP.

  GAPS :

  A policy change towards a targeted, controlled use of antibiotics needs supporting experimental in vitro and in vivo data, including field data from natural outbreaks. In this way, it may be possible to develop a therapeutic regimen that (i) is effective, (ii) minimises transmission, and (iii) minimises the risk of AMR development.

  There is limited in vivo data regarding the efficacy of different antibiotics in reducing clinical disease and transmission and no antimicrobials have been shown to completely clear the infection. The effectiveness of affordable alternative antimicrobial agents must be investigated, as well as that of combined therapies including anti-inflammatory drugs.

  The effect of antibiotic treatments on the development of immunity after recovery and in combination with vaccination must be better assessed (for example when antibiotics are used to treat post-vaccinal reactions).

  Standardised protocols and controls are needed to determine the in vitro MIC values of Mmm to antimicrobials used in the field for the treatment of CBPP and other respiratory syndromes.

  Epidemiological cut-off values (ECOFFs, representing the MIC above which bacterial isolates have phenotypically detectable acquired resistance mechanisms) are not available for animal mycoplasmas. Similarly, clinical breakpoints, relating MIC results to antimicrobial effectiveness in vivo, have not been determined. These cut-offs would help clinical interpretation of MICs and guide towards a more reasonable use of antibiotics. However, determination of ECOFFs under Eucast/Vetcast guidelines will be extremely challenging due to the scarcity of strains available world-wide.

• Future therapy

  The in vitro efficacy of alternative therapies against several mycoplasma species, including Mmc and Mmm, has been explored (Arjoon et al., 2012; Sleha et al., 2014; Furneri et al., 2012; Kama-Kama et al., 2016). Antimicrobial extracts from medicinal plants showed some inhibitory properties in vitro, though their toxicity, pharmacokinetics and pharmacodynamics have not been explored and in vivo efficacy has not been demonstrated. To our knowledge, neither antimicrobial peptides nor bacteriophage therapies have been explored for the treatment of CBPP or other mycoplasmas.

  Antibiotherapy or other means of treatment have not been identified as a priority measure for CBPP control and there are no specific developments for CBPP therapy that we are aware of. Any new molecules will be dedicated for use in human therapy and are unlikely to play a role on CBPP control. On the contrary, there is a global plan to reduce the use of antibiotics in order to reduce AMR, which is one of the greatest health challenges of our times (Murray et al., 2022).

  GAPS :

  The inhibitory activity of antimicrobial peptides and bacteriophages on Mmm should be investigated.

• Commercial potential for pharmaceuticals in Europe

  Not applicable

• Regulatory and/or policy challenges to approval

  Not applicable

• Commercial feasibility (e.g manufacturing)

  Not applicable

• New developments for diagnostic tests

• Requirements for diagnostics development

  CFT is cheap, but is time-consuming and requires skilled staff, as lab implementation and standardisation is not easy. Some reagents are becoming more difficult to obtain (like sheep red blood cells) and have a short shelf life, making it difficult and costly to maintain the diagnostic capacity over time. ELISA is a widespread technique, which can be implemented in many laboratories already running a variety of ELISA tests. CPBB cELISA, commercially available, is more expensive than CFT. However, contrary to CFT, semi-quantitative results are less technician-dependent and can be obtained in less than 3 hours. Both tests need to be conducted in a laboratory.

  The serological responses detected by CFT and cELISA wane over time post-exposure (within 3-4 months for CFT; longer for cELISA), hampering the assessment of chronic infections and asymptomatic carriage. Thus, they must be interpreted at herd level.

  A Swedish research group carried out extensive investigations to identify immunogenic surface proteins and found antibodies against 61 recombinant surface proteins of Mmm in positive bovine sera (Neiman, et al., 2009; Hamsten et al., 2008, 2009 and 2010). They finally used 8 candidates to develop a specific cocktail ELISA. Building on these studies, Jores et al. (2009) carried out further studies on the immunoproteome of CBPP. They identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease presenting severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Based on these and other results, alternative serological detection methods have been developed. Gantelius et al. (2010) published a lateral flow protein microarray based on nitrocellulose membrane spotted with 8 recombinant Mmm proteins. This test is described as a good possibility for implementation of an on-site test to quickly distinguish between healthy and infected animals with a certainty of 97%. The results are available after ~10 min. However, appropriate laboratory equipment is required. A cocktail ELISA and a lateral flow test using three of the above-described recombinant proteins were published by Heller et al. (2016). Therefore, three full length recombinant proteins were expressed in E. coli (not only short peptides) and applied to these two test platforms.

  Other methods did not lead to commercialization (or only temporarily). Bruderer et al. (2002) developed an indirect ELISA using the specific recombinant lipoprotein LppQ. The determined values for specificity and sensitivity were comparable to those of the cELISA. The test was also commercially available for a time. However, production was discontinued after a few years.

  Molecular biology methods are best suited for confirmation of suspicions based on clinical/pathological changes or positive serological results. They are useful at herd level as well as for individual animal studies. Previously described molecular targets that may be used or modified to develop highly specific new technologies (no cross-reactions with the Mmc strain Ker TCR LT, from India) are: (i) the PCR-based test of Bashiruddin et al., (ii) the nested PCR test of Miserez et al., and (iii) the TaqMan real time PCR test of Gorton et al. (see section 1.3).

  In addition to PCR and real-time assays, a microarray platform was developed by Schnee et al. (2012) with the company Abbot. With this platform it was possible to differentiate 83 mycoplasma species. Even mixed infections could be identified. However, it was not possible to differentiate the members of the “Mycoplasma mycoides” cluster. The platform was commercially available for some time from Abbot (Jena, Germany). However, the production of the array was terminated by the manufacturing company due to economic reasons and restructuring within the company.

  There are no suitable commercial pen-side tests available, either for serological diagnosis or for direct detection of the CBPP agent. The production of a commercial LAT was ceased due to its high market price. Other published molecular tests with potential for field use include (i) a direct detection assay based on the LAMP technology (Mair et al., 2013) and (ii) a lateral flow test already developed within a GIZ project for rapid serological screening (Heller et al., 2016), but for which no industrial partner who can produce and validate this test on a larger scale has been identified.

  GAPS :

  Due to limited sensitivity (impossibility to detect very early stages as well as chronic disease), currently available serological tests are not recommended for individual animal diagnosis but at herd level. More sensitive methods are needed for individual diagnosis, particularly for import clearance.

  Also, serological cross-reactions with other mycoplasmas of the “M. mycoides” cluster pose difficulties, especially in eradication programmes using the test and slaughter strategy. Some serological methods lead to false positive results due to these cross-reactions (CFT, LAT). On the other hand, the cELISA is highly specific, though it is not currently available.

  Field validation is required for some molecular diagnostic methods. To our knowledge, none are validated under ISO17025 accreditation. Inter-laboratory assays and/or evaluation by an international expert group would help assess the available assays/kits.

  Development of pen-side tests (for example lateral flow or agglutination tests) could facilitate diagnostics for CBPP outside main laboratories but cost effectiveness will need to be addressed.

  Optimisation of the slide agglutination / LAT should allow the development of a cheap pen-side test for rapid screening in the field in Africa.

• Time to develop new or improved diagnostics

  Time and cost depend on the type of test. In general, the development of tests is much faster and less expensive than developing vaccines. Yest, Development and validation up to commercial availability does usually take several years.

• Cost of developing new or improved diagnostics and their validation

  The development and validation of new tests is time consuming and labour intensive, which is costly. Costs cannot be specified as they will depend on the nature of the test and the cost of producing reagents and supplying reading or processing machines if necessary. Once validated, there will need to be a commercial company willing to market the test, so the involvement of industry in the development and validation must be achieved as soon as possible.

  For PCR, the major factor having both technical and economical constraints is the cold chain required for shipment, highly impacting the test price. Recent work and advances on new technologies (e.g., freeze-drying) might help to overcome these issues

  Uncertainty of market potential and size does not favour the availability of tests, especially Lateral Flow Devices (LFDs), for which the limited market size implies small batches of production for manufacturers, therefore not allowing them to get cheap tests. This is probably not compatible with the market target / Low Income Countries expectation for such products.

• Research requirements for new or improved diagnostics

  For serological tests, the use of recombinant proteins and or peptides is favoured: defining relevant targets (antigens) and test schemes for a highly specific test with good exclusivity (avoiding cross-reactions) while maintaining a good sensitivity, is crucial. Prior studies are therefore needed before development. This has already been addressed for CBPP diagnostics (Neiman, et al. 2009, Hamsten et al. 2008, 2009 and 2010, Jores et al. 2009, Heller et al. 2016).

  For molecular diagnostics, large genomic datasets are needed to allow intra- and inter-specific comparisons. Data are available for Mmm and other members of the “M. mycoides” cluster (whole genome sequences and partial genomic data available from diverse mycoplasma species), which should allow the identification of alternative targets for the design of specific molecular diagnostic tests. These data should however be enlarged, particularly for what concerns other cluster members, which are largely under-represented. Still, the main limitation is the implementation of extensive validation of the diagnostic assays in the field.

  GAPS :

  Extensive comparative genomic analyses conducted on a large sample of strains representing the temporal and spatial diversity of Mmm and the other members of the “M. mycoides” cluster are needed to determine representative pan and core genomes for targeting diagnostic and genotyping tool developments at different levels.

  Many recombinant proteins had been described in the literature (Hamsten et al., 2010). However, critical aspects such as recombinant protein purity are an issue when addressing the test performances, especially specificity.

  Due to strict export, import and biosafety regulations in most countries, the availability of validated sample panels from experimentally and naturally infected animals is very limited.

  However, these are urgently needed for the validation of new tests.

  Market studies and the involvement of manufacturing companies are needed to prioritise and optimise the development of diagnostic inventions into successful commercial products.

• Technology to determine virus freedom in animals

  The monitoring strategy specified in the WOAH Terrestrial Code (Article 11.5.14. to Article 11.5.18.) for determining freedom from disease must be applied (clinical, pathological and serological monitoring of the herds as well as antigen detection in the case of doubtful results).

  Because of the limitations of serological tests, serological surveillance is not the preferred strategy to prove pathogen freedom: Serological test results for CBPP should not be analysed and interpreted individually but in groups of animals from the same herd or epidemiological unit. Both false positive and false negative results may occur in individual animals due to low sensitivity, particularly during incubation in the early stages of the disease (before specific antibodies are produced, which may last for several months), and in the chronic stages of the disease, when antibody titres drop. False-positive results may also occur, particularly by CFT (~2%), of which an important cause is serological cross-reactions with other mycoplasmas, notably other members of the “M. mycoides” cluster. Therefore, the validity of positive/doubtful serological results in CBPP-free countries should be confirmed using either cELISA or immunoblotting test.

  The validity of positive serological results thus needs to be confirmed by post-mortem and bacteriological examination, and serological tests conducted on blood samples taken at the time of slaughter (WOAH Terrestrial Manual, Chapter 3.4.8, Section 2).

  GAPS :

  The CFT and cELISA, recommended for screening and eradication programmes, lack sensitivity and must be interpreted at herd level. The highly specific immunoblotting test is useful as a confirmatory test but is not fit for mass screening (WOAH, 2021).

  Specific and sensitive tools for individual diagnosis as opposed to today’s available tools for diagnosis at herd level would be preferable for reliable import clearance.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  Historically, different live attenuated vaccines were developed and used for disease control worldwide (V5 in Australia, DK32 in Senegal, KH3J in Sudan, and Ben-181 in China) (Thiaucourt et al., 2021). Strain attenuation was obtained by repeated passages on liquid media or using embryonated eggs (T1/44). Overtime, T1/44 and its streptomycin resistant variant T1sr became the recommended CBPP live vaccines and nowadays they are the only Mmm vaccine strains commercially available. A dual vaccine combining live attenuated Mmm (T1sr) and rinderpest virus was successfully applied during rinderpest eradication and greatly contributed to CBPP control in Africa (Thiaucourt et al., 2003). Recently, a bivalent live attenuated vaccine formulation combining T1/44 and LSD virus showed to be safe and immunogenic but protection to in vivo challenge was not assessed (Safini et al., 2022).

  Attempts to develop inactivated vaccines were also reported based either on pleural fluid or Mmm cultures and tested with several adjuvanted formulations. However, this approach was abandoned as live vaccines seemed more efficient and cost-effective (Thiaucourt et al., 2003; Thiaucourt et al., 2021). Data from recent studies indicated 80% protection using a vaccine formulation based on heat-inactivated Mmm administered with Freund’s Complete Adjuvant (Mwirigi et al., 2016a).

  Another study assessed the short-term protection conferred by a formulation based on Mmm-derived capsule polysaccharide (CPS) conjugated to ovalbumin. Booster immunisation with CPS vaccine resulted in reduced pathology (57% efficacy) (Mwirigi et al., 2016b).

  Finally, application of reverse vaccinology led to a prototype vaccine based on Mmm recombinant proteins that elicited protective immune responses against CBPP challenge (Nkando et al., 2016).

  The use of in vivo infection models in cattle is currently required to assess the potency of new or improved vaccines. The in-contact challenge method resembles natural infection, but incubation time and disease outcome can be highly variable (Dedieu et al., 2005). Challenge through endotracheal intubation was proposed as an alternative method to standardise timing of infection and to reduce the number of experimental animals (Nkando et al., 2012, Sacchini et al., 2011). Recently, efforts have been made to develop new infection models based on intranasal atomisation (Sacchini et al., 2020) and nebulisation through a mask (work by VIDO, KALRO and ILRI, unpublished). They have shown improved transmission of classical CBPP in a large proportion of animals, allowing a reduction in the number of animals needed in challenge trials.

  Disease quantification and scoring are critical elements to assess and compare vaccine efficacy. In an attempt to quantitate and integrate the various criteria of animal infection and susceptibility, a scoring system was developed (Hudson & Turner, 1963) and later slightly adapted (Huebschle et al., 2006) to allow vaccine efficacy estimations. Recently, an alternative lung lesion score system was proposed (Di Provvido et al., 2018) but it has not yet been adopted.

  Finally, the development of new tools for Mmm genome editing may bring opportunities for the development of novel vaccines (Ipoutcha et al., 2022).

  GAPS :

  Safer and thermostable vaccines inducing good and long-lasting protection against mortality and clinical disease (at least one year) would improve CBPP control. DIVA vaccines would also be useful in countries implementing eradication plans.

  Multivalent vaccines are needed to dramatically reduce the cost of vaccination in Low- & Middle-Income Countries (LMIC) in Africa. Market studies are needed to develop this strategy.

  The possibility of improving the thermostability of current live vaccines by addition of stabilisers to the freeze-dried formulations must be assessed. Similarly, the potency and duration of immunity may be improved by the use of additives acting as adjuvants enhancing a protective immunity, which has not yet been explored.

  The use of adjuvants in the formulation of new killed or subunit vaccines should also be assessed, ideally following a better understanding of the protective immune response to be elicited.

  Although new tools allowing Mmm genome editing have been developed, no synthetic biology tools are yet available for the manipulation of Mmm genomes in yeast.

  Improved infrastructures and capacity building in vaccine production facilities may be needed to move on from the current live vaccine towards the production of new generation (ex. recombinant and subunit vaccines).

  A reproducible in vivo challenge model is needed to evaluate any current or future vaccines (this is currently under development (VIDO, KALRO and ILRI, unpublished)

  With a more robust challenge model, that is more reproducible and induces more clinical signs and pathology, the current scoring systems need to be re-evaluated.

• Time to develop new or improved vaccines

  New vaccine developments require scaling up, assessing stability, and evaluating the safety, the efficacy and the duration of immunity.

  Developing GMP CBPP vaccine is likely to take 5-6 years, including proof of concept, industrial development, safety and efficacy trials on animals. And this will be similar for improved formulation and for entirely new vaccines.

• Cost of developing new or improved vaccines and their validation

  The cost estimated for the development and validation of an improved quality CBPP vaccine manufactured under GMP conditions using modern technology processes may be of 3.5 M Dollars (rough estimation provided by “MCI Santé Animale").

• Research requirements for new or improved vaccines

  Characterisation of the virulence determinants of the pathogen and, most importantly, of the protective host immune response: little known, as reviewed in Sections “Description of infection & disease in natural hosts > Mechanisms of pathogenicity” and “Detection and Immune response to infection > Mechanism of host response (neutralization, immuno-pathology)”, respectively

  Improvements in the CBPP infection model in cattle may still be required and adapted scoring systems will be needed. Alternative small animal and in vitro models are urgently required for pathogenicity studies, as well as biomarkers correlating with infection and protection in the different models.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  Not applicable

• Time to develop new or improved pharmaceuticals

  Not applicable

• Cost of developing new or improved pharmaceuticals and their validation

  Not applicable

• Research requirements for new or improved pharmaceuticals

  Not applicable.

Disease details

• Description and characteristics

• Pathogen

  Mycoplasma mycoides subsp. mycoides (Mmm), previously known as Mycoplasma mycoides subsp. mycoides "small colony'' biotype (MmmSC), is the etiologic agent of CBPP (Manso-Silvan et al., 2009).

  Mmm belongs to the class Mollicutes, order Mycoplasmatales, family Mycoplasmataceae, and genus Mycoplasma. The Mollicutes, trivially referred to as “mycoplasmas”, are distinguished from other eubacteria by their small size and lack of a cell wall, which renders them intrinsically resistant to beta-lactam and other antibiotics targeting the cell wall. Though technically Gram-negative, mycoplasmas evolved from Gram-positive bacteria by a process of massive genome reduction involving the loss of many metabolic pathways and adaptation to a commensal or parasitic mode of life. This explains their relatively strict host-tissue tropism and fastidious nature in vitro.

  More precisely, Mmm belongs to the “M. mycoides” cluster, a group of five very closely related ruminant pathogens sharing many genetic and phenotypic features (Manso-Silvan et al. 2009).

  Mmm cells are pleomorphic, non-spiral, and non-motile and show a tendency to produce filamentous growth. They are surrounded by an exo-polysaccharide (galactan) forming a pseudo-capsule (capsular polysaccharide, CPS), which is also secreted as a free extracellular form (extracellular polysaccharide, EPS).

• Variability of the disease

  As reviewed in the CBPP chapter of the WOAH manual (WOAH, 2021), The genetic diversity of Mmm strains is relatively well known and Mmm genotyping has been achieved using diverse molecular techniques, including restriction DNA analysis, southern blotting and multi-locus typing, which showed that African and European strains belonged to separate clades and proved that recent European CBPP outbreaks in the 60’s, 80’s and 90’s, were not due to a re-introduction from Africa, but more likely to resurgence from regions from which it had not been completely eliminated (Thiaucourt, 2018).

  Several whole genome sequences are available online, though their comparison is not straightforward because of multiple genome rearrangements and large DNA indels. Furthermore, Mmm is a very monomorphic pathogen so it presents very little intraspecies variability when sequence comparisons are limited to a few loci. To overcome these problems, an extended multi-locus sequence typing (eMLST) approach, based on 62 polymorphic genes of the Mmm core genome, was developed for the fine identification of strains for epidemiological investigations, phylogenetic analysis, and molecular dating (Dupuy et al., 2012). This approach made it possible to estimate the mutation rate of Mmm at 5 x 10^-7 / site / year (for the studied set of genes) and to date the origin of CBPP at around 300 years ago, most likely in Europe, from where it was exported to other continents, including Africa, during the 19th century.

  Mmm strains can be attenuated by serial passage in culture/ animals, which has provided the basis for the development of the live vaccines used today. Strains with diverse levels of pathogenicity are available; notably vaccinal strains with no residual virulence (i.e., type strain PG1^T and vaccine strains T1sr and KH3J) or retaining some (T1/44, V5), though the genetic determinants of virulence and attenuation have not been identified. African and European Mmm strains have been differentiated using diverse genomic methods, indicating that the last outbreaks occurring in Europe at the end of the 20th century were not due to re-introductions from Africa but more likely to resurgence events from regions from which it had not been completely eliminated. Late European strains isolated since 1980 lack a genomic segment containing part of the glycerol import system (Vilei and Fey, 2001), which has been identified as a major virulence determinant in Mmm. These strains have been considered less virulent (Abdo et al., 1998) and reported to cause chronic disease, showing few clinical signs and reduced mortality (Nicholas et al., 1996). However, there is poor evidence for the reduced virulence of Mmm strains, particularly taking into consideration the great variability observed in animal infection trials.

  GAPS :

  Only a few whole genome sequences and partial genomic data of Mmm strains are available and they do not represent the entire temporal and spatial diversity of the species.

  Molecular epidemiology analyses are hampered due to the extreme paucity of CBPP outbreak investigations, sampling and Mmm isolation and characterisation.

  A whole genome analysis pipeline for finest Mmm strain genotyping and comparative genomics and other “omics” analyses should be developed; scripts and data repositories should be made publicly available. This should allow the identification of virulence traits, as well as molecular markers of AMR.

  The genetic determinants of virulence/attenuation and host/tissue tropism need to be investigated among members of the “M. mycoides” cluster, as well as the molecular basis for acquisition of AMR.

• Stability of the agent/pathogen in the environment

  The resistance of the CBPP agent has been described in detail by Provost et al. (1987) and was reviewed in the WOAH card (2020). Due to the lack of a cell wall, mycoplasmas are quite fragile outside their ecological niche. Mmm is very sensitive to physical, chemical, and biological factors and does not persist in the environment for more than 3 days in tropical areas and up to 2 weeks in temperate zones.

  Temperature: Inactivated within 60 minutes at 56°C and 2 minutes at 60°C; pH: Inactivated by acid and alkaline pH; Chemicals/Disinfectants: Inactivated by many of the routinely used disinfectants: mercuric chloride (0.01%/1 minute), phenol (1%/3 minutes), and formaldehyde solution (0.5%/30 seconds). Inactivated by ultraviolet radiation within a few minutes.

• Species involved

• Animal infected/carrier/disease

  Under natural conditions, Mmm affects ruminants of the Bos genus: i.e., mainly taurine (Bos taurus) and zebu (Bos indicus) cattle, and to a minor extent also yak (Bos grunniens) and water buffaloes (Bubalus bubalis).

  Among wild animals, a single anecdotal case was reported in American buffaloes (Bison bison) but none in African buffaloes (Syncerus caffer) or other wild ruminants.

  Host susceptibility has been reviewed by Provost et al. (1987) and Thiaucourt (2018). Cattle of the Bos genus are the most susceptible species to CBPP, whereas water buffaloes are considered less susceptible. There are conflicting opinions regarding possible differences in susceptibility between Bos taurus and Bos indicus cattle breeds, to the disadvantage of the latter, though this has been associated with breeding systems rather than species susceptibility. However, differences in breed susceptibility have been reported, both in regards to natural disease and post-vaccinal reactions (Provost el al., 1987). Furthermore, the susceptibility of individual animals to CBPP infection varies, and a proportion of cattle seem to be naturally resistant to the disease. Age also affects susceptibility, with an initial phase of low susceptibility in calves up to 6 months of age, a subsequent phase of moderate susceptibility gradually increasing up to 12-18 months of age, and a final phase of full susceptibility for cattle over 2 years of age. The reduced susceptibility and atypical presentation of disease in calves may be related to the presence of colostral immunity and to the immaturity of their immune system, though the mechanisms involved in age susceptibility are not totally understood.

  Mmm has been isolated from the lungs of naturally infected goats and sheep, but small ruminants have limited susceptibility and their role in transmission is not considered relevant (see below).

  Animals may carry Mmm in the nasal passages for 40 days during the incubation period, before any serological response can be detected. Also, clinically recovered cattle may develop pulmonary sequestra, which may persist for many months (up to 2 years) and eventually reactivate leading to CBPP transmission. These silent carrier animals may thus disseminate the infection to susceptible animals; they are suspected to play an important role in the spread of the disease, though this has not been demonstrated experimentally.

  GAPS :

  The mechanisms of persistence and transmission from silent carriers (via the reactivation of sequestra lesions) are still unclear.

  The possible carriage of Mmm in alternative sites such as the ear canal, as well as alternative mechanisms of transmission from them, should be studied.

  The possible role of young animals as reservoirs of CBPP within the herd must be elucidated.

• Human infected/disease

  None. Mmm is not a zoonotic agent.

• Vector cyclical/non-cyclical

  None described. However, goat fleas have been identified as vectors for the transmission of the closely related species Mmc, with reproduction of disease in goat kids, and ticks sampled on sheep and goats have been found to carry viable M. agalactiae strains, suggesting a potential role of ectoparasites as reservoirs or vectors of mycoplasma strains (Galluzzo et al., 2021).

  GAPS :

  The possible carriage of Mmm in ectoparasites such as ticks and their possible role in the transmission of CBPP warrant further investigation.

• Reservoir (animal, environment)

  No reservoir has been described and persistence of Mmm in the environment is considered irrelevant given its high sensitivity to physical and chemical stress.

  Mmm has been isolated from sheep and goats in Africa, in Portugal and in India (Srivastava et al., 2000) but neither small ruminants nor wild ungulates are considered to play a role in the epidemiology of the disease. The fact that the disease could be successfully eradicated in Botswana by stamping out the affected and in-contact cattle population, without taking into consideration other ruminant species, seems to confirm this, though specific epidemiological conditions may have been at stake.

  GAPS :

  The possible role of small ruminants (and wild ungulates) as reservoirs of CBPP must be studied.

• Description of infection & disease in natural hosts

• Pathogenic life cycle stages

  None.

• Signs/Morbidity

  Classical CBPP is a strictly respiratory disease characterised by unilateral severe exudative pleuropneumonia. The clinical signs and pathological features of CBPP have been extensively described by Provost et al., (1987) and reviewed by Thiaucourt (2018). Clinical disease may be acute, subacute, or chronic. In the initial stages of an epidemic the disease tends to be acute, but as the epidemic progresses subacute and chronic cases predominate. In endemic areas the disease tends to be mostly chronic and subclinical.

  Acutely affected animals present fever and respiratory distress, characterised by painful breathing and coughing. In severely affected animals, dyspnoea is aggravated by the presence of large volumes of exudate in the thorax and animals then stand with head and neck extended, mouth open, and forelegs wide apart. Death may follow, within one to three weeks in acutely affected animals as a result of respiratory distress.

  Typical acute lesions consist of interstitial pneumonia, characterised by enlarged interlobular septa associated with red and grey hepatisation, which gives a marbled appearance to the lungs, accompanied by severe serofibrinous pleurisy. Most often a single lung is affected. If not pathognomonic, these lesions can be highly evocative of CBPP, though the classical presentation is rarely observed in practice due to insidious disease circulation, antibiotic treatments and concomitant infections.

  In subacute cases the lung lesions are more localised and infrequent coughing may be the only clinical sign. In chronic cases only emaciation and a coughing, provoked when the animal rises or upon exercise, are observed. Severely affected animals that do not die may take many months to recover. However, in many cases the clinical signs gradually disappear, after which the general condition of the animals improves rapidly.

  In the chronic form / stage lesions resolve leading to the formation of characteristic encapsulated necrotic lesions (or sequestra), in which Mmm may remain viable for months. The pleural fluid disappears and pleural adhesions are prominent.

  In calves up to six months the only sign of disease is usually arthritis, particularly of the carpal and tarsal joints. Pulmonary lesions may also develop occasionally.

  It must be noted that Mmm can also induce severe lesions when inoculated artificially by other routes. This is the case of the invasive oedematous lesion, known as Willems’ reaction, that is observed at the site of injection when Mmm is inoculated subcutaneously. These local reactions, that may occasionally develop upon vaccination with strain T1/44, can evolve into systemic disease and may even result in the death of the animal.

• Incubation period

  Very variable, from 2 weeks to 6 months, with an estimated mean between 4 and 6 weeks (Thiaucourt, 2018). Mmm excretion can occur during this period for up to 40 days (Hudson and Turner, 1963), while animals do not display any symptoms and no serological response can be detected.

  GAPS :

  A better understanding of the factors influencing the duration of the CBPP incubation period is needed.

• Mortality

  In fully susceptible herds, upon a first introduction, mortality may be as high as 80% in the absence of antibiotic treatments. When CBPP was reintroduced in the Western Province of Zambia in 1969, this outbreak resulted in 75% morbidity and up to 68% mortality (Thiaucourt et al., 2018). Experimental infection trials in Australia resulted in mortalities varying from 50 to 90% (Provost et al., 1987).

  GAPS :

  Mechanisms that make some cattle succumb to disease, while others withstand the disease challenge and go through the whole course up to chronic levels and, eventually, recovery are not well understood. Biomarkers of disease severity may be useful for prognosis. This understanding is important also for modelling.

• Shedding kinetic patterns

  Shedding is maximal during the acute stage of the disease (around 15 days post-infection), when animals cough, excreting infected droplets. It appears that animals in the chronic stage of the disease can still excrete mycoplasmas up to 2 years post-infection, although neither the intensity and periodicity of shedding nor the factors precipitating it are understood.

  GAPS :

  Precipitants and patterns of Mmm shedding from sequestra and any other alternative sources need further investigation.

• Mechanism of pathogenicity

  CBPP is characterised by an exacerbated inflammatory reaction that may be induced directly by toxic mycoplasma components or indirectly, through the release of pro-inflammatory cytokines. No classical virulence factors such as adhesins or toxins have been identified in Mmm genomes, although certain molecules have been shown to be involved in cell adhesion. Virulence has been mainly attributed to surface or secreted components and intrinsic metabolic functions.

  CBPP is the result of an overwhelming inflammatory response leading to tissue destruction and respiratory distress. The precise mechanisms underlying the inflammatory response are unclear but Mmm strains with different levels of virulence in subcutaneous models are available (avirulent PG1 & KH3J, vs. intermediate strains T1/44 and V5, vs. virulent isolates). Either multiple elements or a cascade induced via the activation of immune and somatic cells instead of a single key Mmm factor are more likely to be involved in CBPP pathogenicity, notably in the activation of the exacerbated inflammatory response.

  There are multiple factors produced or triggered by Mmm that have been proposed as potential initiators of inflammation (reviewed by Di Teodoro et al., 2020). These include: i) hydrogen peroxide (H2O2) produced via the metabolism of glycerol that can induce direct cytotoxicity and indirect tissue damage via neutrophil activation. The L-a-glycerophosphate oxidase (GlpO) enzyme was thought to be responsible for the production and release of H2O2 but the recent report of its strict cytoplasmic location implies that Mmm exports the generated H2O2 through other mechanisms to minimise self-toxicity (Schumacher et al., 2019). However, direct cytotoxicity is not observed in pulmonary explants and comparable H2O2 amounts are produced by virulent and intermediate strains. Finally, studies using Mycoplasma gallisepticum mutants lacking GlpO and incapable of producing H2O2 indicated that this enzyme had no impact on lesions scores in vivo (Szczepanek, 2014); ii) lipoproteins, which are potent stimulators of macrophages (Muhlradt et al., 1998); iii) a lipid-anchored polysaccharide termed galactan, although this was inferred from old in vivo studies using partially purified galactan, so its role in triggering an inflammatory response needs to be demonstrated. A genetic on/off switch of the ptsG gene involved in glucose metabolism is responsible for the alternate expression of capsular and free extracellular galactan (Gaurivaud et al., 2004; Bertin et al., 2013). Differences in resistance to host defence mechanisms in capsulated versus non-capsulated Mmm variants were observed (Gaurivaud et al., 2014). Furthermore, non-capsulated forms could make pro-inflammatory lipoproteins more accessible, thus triggering or exacerbating inflammation. Finally, a possible role for capsular galactan in autophagy-mediated inflammation cannot be ruled out (Ding et al., 2019); iv) pro-inflammatory cytokines released by phagocytes; v) secreted proteases (Ganter et al., 2019); vi) a two-protein system (MIB-MIP) capable of capturing and cleaving the VH domain of immunoglobulins, rendering cryptic epitopes accessible to recognition by phagocytes, as demonstrated in Mmc (Arfi et al., 2021). Subsequently, oxidative burst and release of IL-8, a well-known potentiator of neutrophil recruitment, are triggered. However, the MIB-MIP system is widely present in many distant Mycoplasma spp. including non-pathogenic strains.

  Mechanisms allowing Mmm to evade the immune response and resist elimination by the host include: i) phase-variation surface antigens (Persson et al., 2002); ii) immunoglobulin-blocking proteins (MIB-MIP system; Arfi et al., 2016); iii) biofilm formation (McAuliffe et al., 2008); iv) adhesion to epithelial cells that may prevent elimination by phagocytes (Di Teodoro et al., 2020); v) toxin-antitoxin systems (Hill et al., 2021); vi) free extracellular galactan detected in circulating blood during acute CBPP may dampen immune defences (Rodrigues et al., 2015) through its anti-inflammatory properties (Totté et al., 2015); vii) capsular galactan (Jores et al., 2019).

  A more comprehensive understanding of the genetic determinants of host/tissue specificity and virulence of Mmm and other mycoplasmas of the “M. mycoides” cluster, as well as the regulation of their expression, may be obtained from the analysis of comprehensive whole genome data. Very few whole genome sequences are available to date and the achievement of complete circularised genomes in these mycoplasma species is hampered by the presence of numerous insertion sequences, integrative conjugative elements, and large duplications. However, this may now be facilitated by the improvement and democratisation of the most recent sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technology (ONT) platforms, which can now generate long reads (thousands of base pairs) with high accuracy. This will pave the way for other “omics” analysis, leading to the identification of relevant virulence factors.

  Other recent developments that bring new opportunities to CBPP pathogenicity studies are: (i) genome editing tools allowing the generation of Mmm targeted mutants for functional analyses (Ipoutcha et al., 2022); (ii) expression systems for the production of intrinsically fluorescent Mmm strains, which facilitate the analysis of host-mycoplasma interactions in vitro and in vivo (Tiffany et al., 2016); (iii) new developments in infection models for the study of such interactions.

  Infection models are used to study the pathogenesis and assess virulence determinants, notably through the analysis of targeted mutants. In vitro infection models used so far to study cytotoxicity and adhesion include bovine cell lines such as nasal and lung epithelial, aorta endothelial, skin fibroblasts (Bischop et al., 2008; Aye et al., 2015), and 3D systems including bovine respiratory explants (Di Teodoro et al., 2018) and bovine precision-cut lung slices (PCLS, Weldearegay et al., 2019). Interactions with phagocytes was studied in vitro using ex vivo primary cultures of monocytes-derived macrophages (Bonnefois et al., 2016), alveolar macrophages (Jungi et al., 1996), and polymorphic nuclear cells (Di Teodoro et al., 2018; Di Federico et al. 2020).

  Regarding in vivo infection, no small animal model exists, so assays must be performed in cattle. The in-contact model of infection requires large numbers of animals and the incubation time can be highly variable. Endo-bronchial intubation generally leads to sub-acute rather than acute CBPP and is not very reproducible. However, efforts to develop novel infection models using aerosolized Mmm have been undertaken recently (Sacchini et al., 2020; VIDO, KALRO, ILRI unpublished) and are likely to promote the study of pathogenesis in vivo.

  In an attempt to quantitate and integrate the various criteria of animal infection and susceptibility, a scoring system was developed (Hudson & Turner, 1963) and later slightly adapted (Huebschle et al., 2006) to allow vaccine efficacy estimations. Recently, an alternative lung lesion score system was proposed (Di Provvido et al., 2018) but it has not yet been adopted.

  GAPS :

  A clear picture of the molecular basis of Mmm’s virulence and attenuation is lacking and research is needed to understand how the host’s environment affects the regulation of Mmm virulence genes.

  Particularly, the early mechanisms leading to exacerbated inflammation and infection need to be elucidated, as well as those driving Mmm persistence in the host.

  Although the mechanisms leading to the generation of H2O2 are well described, it remains unclear how H2O2 is excreted. Furthermore, a direct cytotoxic effect of Mmm via H2O2 is observed in vitro but only at high Mmm concentrations, which questions its real impact in vivo in the early stages of CBPP.

  The precise role of galactan (free vs. capsular form) in triggering inflammation on one side and immune-suppression and evasion on the other is unclear. Interactions of capsulated and non-capsulated Mmm variants with phagocytes should be investigated to address this.

  The capacity of epithelial and endothelial cells to produce pro-inflammatory cytokines/chemokines in response to Mmm has not been investigated. Moreover, Mmm antigens, such as lipoproteins, triggering the production of pro-inflammatory cytokines remain to be identified.

  The functionality of the MIB-MIP system in attenuated and avirulent strains has not been investigated. Its possible role as a trigger of inflammation in CBPP must be elucidated.

  Very little is known about the role of Mmm’s extracellular secretome, including secreted proteases, in pathogenicity.

  It is not clear how Mmm survives in sequestra despite tissue destruction and other possible mechanisms underlying Mmm’s persistence in and outside the host need to be unravelled (e.g., role of biofilms).

  Targeted mutant studies would facilitate the understanding of virulence mechanisms in CBPP pathogenicity, starting with in vitro models. There is much room for improvement of experimental infection models both in vivo and in vitro.

  More comprehensive in vitro models, such as respiratory explants and PCLS and multicellular co-cultures (e.g., epithelial/endothelial cells mixed with phagocytes), could also be very useful to compare strains of diverse virulence and mutants.

  A reproducible in vivo challenge model is needed to further evaluate targeted mutants. Novel techniques allowing the use of aerosolized inoculums should be investigated in order to reproduce more closely the natural infection. Also, the “Willems’ reaction” model could be used to analyse the impact of targeted Mmm gene deletion on inflammation in vivo.

  Finally, although a number of virulence factors have already been identified, more comprehensive “omics” analyses on “M. mycoides” cluster strains of diverse host and tissue tropism, as well as virulence, may lead to the identification and/or selection of relevant virulence factors for further functional studies.

• Zoonotic potential

• Reported incidence in humans

  None.

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

  None.

• Symptoms described in humans

  None.

• Likelihood of spread in humans

  None.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Very high impact on animal welfare. Severe pain in breathing, due to pulmonary pathology, and heavy mortality in the acute form of the disease. Emaciation in chronic stages. Occasionally, post-vaccinal reactions can also induce pain and even lead to death if untreated.

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

  None.

• Slaughter necessity according to EU rules or other regions

  Slaughter is necessary to control the disease when it occurs depending on the epidemiological situation. In disease-free zones/countries, stamping out or slaughtering of infected and in-contact animals is necessary. Slaughter of animals may lead to potential loss of useful genetic resources (e.g., indigenous breeds with precious attributes such as trypanotolerance).

• Geographical distribution and spread

• Current occurence/distribution

  The occurrence of CBPP is summarised in the WOAH CBPP card (WOAH, 2020). The WOAH has a procedure for the recognition of CBPP-free countries/areas :

  http://www.oie.int/en/animal-health-in-the-world/official-disease-status/

  The detailed information on the occurrence of CBPP may be obtained from the WOAH database interface (WAHIS) :

  http://www.oie.int/wahis/public.php?page=home

  CBPP is confined to the areas south of the Sahara Desert, from the Tropic of Cancer in the north to the Tropic of Capricorn in the south (Amanfu, 2019). The Asian situation is unclear, due to lack of efficient surveillance (with the exception of China, where CBPP has been eradicated). In Europe CBPP has not been reported since the last case detected in Portugal in 1999.

  New areas are being invaded as observed in Senegal and Gambia in West Africa in 2012. Most countries in Southern Africa are free from the disease and all efforts should be made to stop contamination of this region.

  GAPS :

  The true distribution and incidence of CBPP should be identified in more detail, since disease reporting to WOAH is very limited and so underestimates occurrence.

  The current situation of CBPP in Asia should be clarified (for example, positive CBPP serology has been reported in Malaysia, though the disease has not been officially confirmed by specific identification of the agent).

  Improved disease reporting will require efforts to increase awareness and capacity building in regards to CBPP surveillance.

• Epizootic/endemic- if epidemic frequency of outbreaks

  Endemic in most countries of sub-Saharan Africa. Characterised with episodes of high clinical disease and quiescence. Disease usually rekindles in herds that had earlier episodes after some time. There is no general pattern for this re-occurrence. Sporadic epizooties have been recently recorded in protected areas in North Namibia and Zambia due to the uncontrolled introduction of infected animals.

  GAPS :

  At herd level, the relation between demographic/seasonal trends and temporal patterns of recurrence should be investigated in more detail.

• Speed of spatial spread during an outbreak

  Outbreaks often occur as a result of cattle movement leading to contact between infected and naive animals, and cattle movement has long been identified as the most important factor in the spread of the disease.

  Since Mmm cells do not persist in the environment for long periods of time the possibility of indirect transmission through fomites or animal products can be practically excluded.

  The speed of spatial spread thus depends on the frequency and intensity of movement of infected cattle. If well restricted, the infection can be contained within the outbreak zone without spreading. The presence of silent infections during incubation and chronic carrier stages is difficult to monitor and leads to new foci. Close contact between infected and susceptible herds is considered an essential prerequisite for sustained spread of the disease.

  GAPS :

  Difficulties to estimate spatial spread are related to animal mobility, in particular asymptomatic ones. Rough estimates of speed and spatial spread could be provided once these data are collected:

  • Animal movements, origin and destination at national and international level, number of heads, together with transportation. These data could identify possible hubs and main axes as well as the average travel time of animals.
  • Outbreak declarations (with geographical information and time stamp) that could be used for spatial statistical analysis

  More understanding on the environmental persistence (and transmissibility) of the pathogen should be achieved, since days or weeks of persistence could result in long range, international spread.

  Since livestock movements can involve different species at the same time, the role of other potential reservoirs such as small ruminants should be better assessed.

• Transboundary potential of the disease

  The transboundary potential of CBPP is very high, especially where there is exchange/movement of live cattle. CBPP is a classic example of a transboundary animal disease whose control often requires coordinated efforts between countries.

  Trade data are collected by the UN Comtrade database platform (https://comtradeplus.un.org/) and need to be explored to give a representation of the spatial and temporal patterns.

  GAPS :

  Data on transboundary movements in Africa are rarely collected, not centralised and not harmonised. No information is available regarding illegal animal movements.

  Trade data collected by commercial and trade platforms need to be explored to give a representation of the temporal patterns of animal movements to assess the risk of introduction into Europe, notably via introduction from Africa to the Middle East and then Europe.

• Route of Transmission

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

  The modes of transmission of CBPP have been reviewed by Provost et al. (1987) and are listed in the CBPP card (WOAH, 2020). The disease is usually transmitted by the airborne route, via the inhalation of infective droplets excreted through coughing and sneezing.

  As previously stated in section 8.1, chronically infected animals may present sequestered lesions that can reactivate leading to CBPP transmission. These silent carrier animals are suspected to play an important role in the spread of the disease, though there is no experimental evidence supporting this.

• Occasional mode of transmission

  More anecdotal sources and routes of transmission have also been reported (Provost et al., 1987; WOAH, 2020). Airborne transmission at a distance of up to 200 metres may occur under favourable climatic conditions. Also, transient bacteraemia during the early febrile stage of the disease may render other organs infective. Mmm may thus be present in the saliva, urine, foetal membranes and uterine discharges from acutely affected cattle, and may also be transmitted through aerosols generated from these alternative sources. Transplacental infection may also occur and there are a few anecdotal reports of transmission on fomites, although mycoplasmas do not survive for long periods in the environment and indirect transmission is thought to be unimportant. Mmm has also been isolated from bull semen, but transmission through semen requires further investigation.

  GAPS :

  The risk of Mmm transmission from urine and any body fluids of infected cattle other than the respiratory tract should be assessed, as well as alternative routes of infection.

• Conditions that favour spread

  The common practices of kraaling animals at night and congregating large numbers of animals in a location, as is observed at markets and watering and grazing points, greatly facilitate infection. Stress induced through cattle movement to such locations, exacerbates the spread of the disease.

• Detection and Immune response to infection

• Mechanism of host response

  As expected, given the local inflammatory response characteristic of CBPP, phagocytes containing Mmm antigens as well as lymphocytes are observed in infected lungs (Di Teodoro, 2020). The pro-inflammatory cytokines IL-1β, IL-17A, and TNF-α are detected in vivo in infected pulmonary tissues but the cellular source has not been characterised (Sterner-Kock et al., 2016). Interestingly, IL-17A plays a pivotal role in neutrophil-mediated exacerbated inflammation in general and Mycoplasma pulmonis infection in particular (Mize et al., 2018). TNF-α and IL-1β, are produced in vitro in response to Mmm by alveolar macrophages and neutrophils respectively (Jungi et al., 1996; Di Federico et al., 2020). The respiratory burst of neutrophils is greatly enhanced by Mmm in vitro in the presence of glycerol (Di Teodoro et al., 2018).

  There is very limited data on innate defence mechanisms to CBPP, most likely due to a large consensus regarding mycoplasma resistance to phagocytosis and killing by neutrophils and macrophages in the absence of specific antiserum (Marshall et al,1995). This was recently confirmed for Mmm, even in conditions of non-specific opsonisation by complement (Totté et al, 2023). The same study suggested a potential role of complement in resistance to CBPP through direct bactericidal activity.

  Both humoral and cell-mediated immune responses are detected after infection with Mmm. There is no direct correlation between titres of different immunoglobulin isotypes and either disease severity or protection (reviewed by Di Teodoro et al., 2020) except for IgA, for which a positive correlation with reduced pathology is observed (Niang et al., 2006). Antibody transfer experiments are limited and inconclusive. However, a monoclonal IgM antibody directed against a carbohydrate epitope inhibits the growth of Mmm in vitro in the absence of phagocytes and complement, which is the basis for the growth inhibition diagnostic assay (Kiarie et al., 1996). Immunisation against strongly immunogenic surface-exposed Mmm antigens such as LppQ can induce complications rather than protection upon challenge (Mulongo et al., 2015).

  Strong Mmm-specific memory CD4+ T-cell responses are present in convalescent animals and after full recovery from CBPP (Totté et al., 2008) and are progressively induced after several rounds of vaccination (Totté et al. 2013). This is not surprising given the extracellular localization of Mmm, which promotes MHC Class II-dependent antigen presentation. Nevertheless, in ruminants CD4+T cells are required for strong antibody responses and can amplify phagocytosis through the release of IFN-γ (Goddeeris et al., 1994). Depletion studies in vivo suggest that CD4+ T cells do not contribute to either resistance or pathology during a primary infection (Sacchini et al., 2011). However, this does not exclude a possible role of CD4+ T-cells in protection against a secondary infection.

  GAPS :

  The precise contribution of different immunoglobulin isotypes to protective immunity and immuno-pathology is unclear. Transfer experiments should be performed using improved nebulisation-based challenge methods allowing a reduction of animal numbers. Emphasis should be put on IgAs, given their importance in the mucosal environment. Finally, the capacity of antiserum from convalescent animals to induce killing of Mmm should be confirmed using alveolar macrophages and neutrophils.

  Similarly, there is a lack of understanding of the contribution of cell-mediated immunity in both pathology and protection. The lack of a laboratory animal model for CBPP is a major hurdle to unravel the contribution of the different lymphocytes.

  Concerning CBPP pathogenesis, more in vitro studies on interactions of phagocytes with Mmm strains of different virulence are needed to further explore the interplay of chemokines/cytokines involved. In particular, identifying the host cell source of IL-17A and Mmm antigens that trigger its production may be important. Finally, the in vivo CBPP infection model based on the “Willems’ reaction” could be used to evaluate the effect of neutralising antibodies against chemokines/cytokines, such as IL-17A.

  Regarding protection, it is commonly accepted that animals that fully recover from the disease are protected for life, but this has not been demonstrated experimentally. Moreover, whether full protection (i.e., in the absence of sequestra) can be achieved or not is not known.

  Genomic and immunological tools should be used to identify bio-signatures and correlates of resistance to primary and secondary infection. However, experimental infection / immunisation protocols leading to full protection against reinfection are needed. Indeed, the ultimate goal is to characterise protective immunity leading to full recovery rather than chronic infection.

• Immunological basis of diagnosis

  Antibodies against Mycoplasma mycoides subsp. mycoides are used for diagnosis.

  Classical direct diagnostic tests based on the use of specific antisera (primarily growth inhibition test, immunofluorescence and dot immunoblotting) were necessary for the identification of the CBPP agent in culture before the advent of molecular diagnostic techniques.

  The main serological tests used for CBPP surveillance are the cELISA and the CFT. The CFT affords earlier detection (several days) than the cELISA but it also becomes negative earlier. Detection by cELISA is delayed but remains positive for a longer period after infection (Schubert et al. 2011). This is a consequence of the different immunoglobulin classes covered by each method. Antibodies of the IgG class have a greater affinity in the cELISA, while IgG2 subclass antibodies are unable to fix complement, used in the CFT. On the other hand, IgM class antibodies, characteristic for early infection, are complement fixing and thus readily detected by CFT. This can explain the earlier detection of antibodies by CFT.

  Similarly, antibodies of the IgM class are agglutinating and thus rapid tests based on serum agglutination such as the LAT are most suited for early detection at the pen-side.

• Main means of prevention, detection and control

• Sanitary measures

  The most effective control method that has been successfully used for CBPP eradication (i.e., in the USA and the UK in the late 19^th and early 20^th century; more recently in Botswana) is the drastic stamping out or slaughter of affected and in-contact cattle.

  In case of an outbreak in Europe and other CBPP-free countries stamping out of the animals in the affected establishments and, restrictions of animal movements need to be implemented, with backward and forward traceability and surveillance activities within and outside the restricted zones (EFSA AHAW Panel, 2022).

  In countries where the disease is endemic, drastic stamping out strategies cannot be implemented due to socioeconomic factors, since governments do not have the resources to compensate farmers to allow cattle to be replaced. More importantly, stamping out is not useful if strict livestock movement restrictions cannot be imposed to prevent the reintroduction of the disease.

• Mechanical and biological control

  None (no relevant environmental persistence and mechanical or vector-borne transmission demonstrated to justify this).

• Prevention through breeding

  Host species susceptibility is debated (zebu versus taurine breeds) due to non-consensual evidence. However, it is generally accepted that certain breeds are more susceptible than others (Provost et al., 1987) and differences in individual animal susceptibility have also been observed. A percentage of animals in a herd (typically 10-15%) appears to be naturally resistant to the disease, though this may not be exclusively dependent on genetic determinants, since many other factors may impact the immune response of the animal. Identification of the genetic determinants of resistance to CBPP infection may eventually lead to the selection of resistant animals through breeding, though the interest of such strategy is debatable.

  GAPS :

  The genetic determinants of resistance to CBPP infection have not been characterised.

• Diagnostic tools

  Periodic surveillance that involves abattoir or slaughter slab inspection of lungs and respiratory adnexa, followed by rapid diagnostic testing and cattle movement management are all essential procedures to allow a rapid response and maintain a CBPP-free status. For this reason, rapid molecular diagnostic methods are used upon clinical/pathological suspicion in European, American and some Asian countries. CFT, cELISA and Immunoblotting are also used for animal testing before trade. According to the CBPP manual (WOAH, 2021), CFT and cELISA are recommended for screening and eradication programmes, while the highly specific immunoblotting test is useful as a confirmatory test but is not fit for mass screening.

  In African countries where CBBP is endemic, findings from the slaughterhouse are primarily used for diagnosis. In addition, clinical diagnosis is the main focus in live animals. Direct pathogen detection by isolation of Mmm and molecular detection methods play only a minor role in these countries and pen-side tests are not available for rapid diagnosis in the field. CFT and cELISA are occasionally used for diagnosis and for prevalence studies.

• Vaccines

  Attenuated vaccines that are available on the market reduce disease occurrence and morbidity but can neither prevent the infection nor the development of pathological lung lesions, and thus disease transmission.

  CBPP vaccination campaigns conducted in Africa in recent years have not covered a large proportion of the cattle population. The estimated annual production of CBPP vaccine in Africa over the period 2015 to 2016 was 40-50 million doses, which represented around 10% of the total cattle population at risk. (Thiaucourt, 2018). In addition, low protection rates have been afforded by the vaccine, probably related to factors such as poor handling and administration methods.

  Vaccination campaigns should take into consideration both patterns of seasonal livestock movements and demographic dynamics in order to maximise efficacy.

  Vaccination alone is not sufficient to eradicate the disease, especially in areas where uncontrolled movement of cattle takes place. It is therefore assumed that strategies combining different control tools will be needed to eradicate CBPP (Mariner et al., 2006). Several alternative combinations may be proposed but they need to be validated.

  GAPS :

  The efficacy of CBPP eradication strategies combining different control measures should urgently be validated by performing controlled field studies.

  Disease transmissibility from vaccinated and subsequently infected animals needs to be better assessed, as well as shedding levels. This also applies to animals treated with antibiotics, which are considered potential carriers.

• Therapeutics

  Although there are few reports on the use of antibiotics for the control of CBPP in endemic areas, it is well known that farmers use tetracyclines, but also macrolides and quinolones, to treat clinically infected cattle in order to reduce both mortality and transmission (Thiaucourt, 2018). However, antibiotic treatments do not clear the infection and recovered animals may become potential carriers. Furthermore, antibiotics are freely available in most countries and they are used with little or no antibiotic stewardship (Caudell et al., 2017; Caudell et al., 2022). The quality of the antimicrobials, as well as the optimal dose and duration of treatments is not guaranteed, while withdrawal periods are not respected to ensure safety and limit the development of AMR. Antimicrobials are also used in the case of post-vaccinal reactions to prevent vaccine rejection by farmers.

  No antibiotic-resistant Mmm strains have been reported to date. However, an increasing emergence of resistant M. bovis strains has been reported world-wide over the last few decades, as indicated both by reports of treatment failure and by the identification of genetic mutations consistent with AMR (Lysnyansky and Ayling, 2016). Lack of antibiotic stewardship is likely to lead to the acquisition of AMR also in Mmm strains.

  For these reasons, the use of antibiotics is officially banned in most countries and has been strongly discouraged by WOAH and FAO for many years. However, the possibility of changing the policy on CBPP control to permit the use of antibiotics during outbreaks has been under debate for a long time (Amanfu, 2006; Mariner et al., 2006; Ssematimba et al., 2015).

  GAPS :

  There is little qualitative and quantitative information on the use of antibiotics in the field in Africa. Data on antibiotics used, protocols, and efficacy of treatments are lacking.

  We are not aware of reports of treatment failure and there is no data available regarding acquired AMR by Mmm strains in the field

  There is an urgent need to provide guidelines to optimise antibiotic use and promote antibiotic stewardship (molecules, dose and duration of treatment, as well as withdrawal periods) in order to minimise both the side effects and the development of AMR.

  Furthermore, combined therapies including anti-inflammatory drugs should be explored to further reduce mortality, clinical signs and lesions.

  The strategic and targeted use of antibiotic therapy as a tool in integrated programmes for the eradication of CBPP deserves further investigation (Thiaucourt, 2018). The efficacy of control strategies including targeted antibiotic treatments combined with mass vaccination and slaughter must be evaluated under controlled conditions before this approach can be formally recommended for the eradication of CBPP.

• Biosecurity measures effective as a preventive measure

  CBPP generally requires close and repeated contact between infected and susceptible animals. Biosecurity measures are thus directed at separating the two subpopulations.

• Border/trade/movement control sufficient for control

  CBPP is basically a disease of cattle movements. In CBPP-free countries and regions a ban on the importation of live animals from countries where CBPP is present is the most effective measure to maintain the disease-free status. Official trade is easy to control but illegal movements of animals constitute the main risk of introduction.

  As stated elsewhere, international cooperation is key to managing the transboundary spread of CBPP disease through movement controls. Porous international borders in sub-Saharan Africa make the control measures difficult to implement.

  GAPS :

  Measures should be put in place to monitor animal movements between zones in a country and between international borders. However, livestock movement monitoring and control is very difficult to achieve due to lack of animal identification systems in many countries in Africa. More importantly, restriction of animal movements in regions that practise extensive nomadic and transhumant livestock herding is extremely difficult.

  The net effects of cross border movements are not quantified. This is a big gap.

• Prevention tools

  None.

• Surveillance

  Well planned and coordinated field and abattoir surveillance is paramount for detecting disease and gauging control through stamping out/slaughter, restriction of animal movements, treatments and/or vaccination, according to the disease status and selected control strategies.

  Abattoir surveillance is effective in detecting lesions and can be used to trace back to the herd of origin, though laboratory processing of samples is rarely implemented in Africa and traceability is almost impossible. Serological surveillance, interpreted at herd level, assists in early detection of disease, for prevalence studies aiming at orienting and assessing the efficacy of vaccination campaigns and for international trade.

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

  Historical examples of successful CBPP eradications have been reviewed by Thiaucourt (2018). A strict slaughter protocol was used to eradicate the disease in the USA and the UK at the end of the 19th century, when modern diagnostic tools were not available. More recently, a drastic slaughter policy was also successfully applied in Botswana, which required the strict control of animal movements to prevent the reintroduction of the disease and a great investment for compensating farmers and for restocking purposes. In Australia, eradication was achieved using a strategy combining vaccination and stamping-out and supported by implementation of strict control of animal movements. The disease prevalence was first greatly reduced by applying successive rounds (every 3 months) of serological testing and elimination of positive animals, accompanied by vaccination of all other animals. Then vaccination was discontinued and complete eradication was achieved by slaughtering the remaining infected herds. More recently, Italy and Portugal regained CBPP-freedom by applying a strict test and slaughter strategy.

• Costs of above measures

  Cost of control varies and is influenced by a number of factors. Funds are mostly for testing and logistics, where compensation is waived through sale of carcasses to abattoirs, which put the meat in the food chain after inspection.

  In Botswana, costs of eradication of CBPP in 1995 were estimated at USD 97.5 million, more than the USD 86 million generated in export revenues in 1998 (Mullins et al. 2000). These costs include movement controls, depopulation, compensation, and restocking costs.

  In Narok District of Kenya, annual costs of disease response mitigation were estimated at USD 183,000 (Kairu-Wanyoike et al., 2017). These costs included reporting costs, treatment of sick animals and those with adverse reactions to treatment, sampling/testing, ring vaccination, and deaths due to vaccination or abortions.

  Onono et al., (2014) estimated a strategy of preventive vaccination against CBPP in Kenya to be USD 193/100 cows per year. Slaughter of diseased animals was estimated at USD 912/100 cows per year.

  GAPS :

  The actual cost of control is not known, aside from the case studies provided.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  Yes.

• WOAH disease card available

  https://www.woah.org/app/uploads/2021/03/cbpp.pdf

  GAPS :

  Not applicable.

• WOAH Terrestrial Animal Health Code

  WOAH Terrestrial Animal Health Code 2022. Chapter 11.5. Contagious Bovine Pleuropneumonia. Last update 2014.

  https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&L=1&htmfile=chapitre_mycoplasma_mycoides.htm

  GAPS :

  Not applicable.

• WOAH Terrestrial Manual

  WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2022. Chapter 3.8.4. Contagious Bovine Pleuropneumonia. Last update 2021.

  https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.04.08_CBPP.pdf

  GAPS :

  Not applicable.

• Socio-economic impact

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

  None.

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

  None.

• Direct impact (a) on production

  Mortality may be as high as 80%, especially in naive cattle populations. Productivity loss in endemic areas is marked. Use of oxen power in agriculture is severely impaired during outbreaks of the disease due to decreased ploughing of farm lands and transport of farm produce. Also, use of cattle in social events such as marriages and dowry payment are affected.

  Masiga et al. (1996) estimated direct and indirect costs of CBPP in the 27 countries in Africa in which it occurs at USD 2 billion. Tambi et al. (2006), in a sample of 12 African countries, estimated the direct costs of CBPP at Euro 30 million per year (mortality and morbidity). Production losses in Narok district of Kenya were estimated at USD 1.62 million per year (Kairu-Wanyoike et al., 2017). Onono et al. (2014) estimated the direct annual costs of CBPP to Kenya at USD 2.1 million per year.

  GAPS :

  The exact cost of losses at primary production stages such as farm levels has not been well quantified across a wide evidence base at national and global levels. A few increasingly dated studies exist for Africa, but no prospective impact studies of non-African contexts were identified.

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

  Significant, especially to public services. Control measures are mostly done as a government good. The stamping out method of control leads to loss of valuable genetic resource base (e.g., trypanotolerance in N’Dama cattle of West and Central Africa).

  In Botswana, costs of eradication of CBPP in 1995 were estimated at USD 97.5 million, more than the USD 86 million generated in export revenues in 1998 (Mullins et al., 2000). These costs include movement controls, depopulation, compensation, and restocking costs.

  In Narok District of Kenya, annual costs of disease response mitigation were estimated at USD 183,000 (Kairu-Wanyoike et al., 2017). These costs included reporting costs, treatment of sick animals and those with adverse reactions to treatment, sampling/testing, ring vaccination, and deaths due to vaccination or abortions.

  Onono et al. (2014) estimated a strategy of preventive vaccination against CBPP in Kenya to be USD 193/100 cows per year. Slaughter of diseased animals was estimated at USD 912/100 cows per year.

  GAPS :

  Estimates of direct impacts at primary production stages such as farm levels have not been well quantified across a wide evidence base at national and global levels. A few increasingly dated studies exist for Africa, but no prospective impact studies of non-African contexts were identified.

• Indirect impact

  Very significant, especially where live cattle trade is prohibited. Price depression of cattle in such areas is noticed. Conversely, outbreaks can reduce the availability of meat, contributing to local inflation (Muuka et al., 2012). Outbreaks can also affect milk production leading to protein deficiency, as well as crop production where oxen are used for land preparation and traction of farm produce, since their use is affected due to bans on cattle movements. Haulage of agricultural commodities and ploughing are commonly affected (Wanthanji et al., 2015). Such food insecurity may in the long-term lead to loss of societal cohesion.

  The use of cattle in social events such as marriages and dowry payments is also affected due to the prohibition of cattle movement. The social status may also be impacted. Wealth in many rural societies in Africa is measured by the number of cattle owned by ‘peasant’ cattle farmers. Loss of societal standing thus results from the direct effect of the disease, as well as the adoption of control strategies leading to a reduction in the cattle population in the absence of compensation or other relevant replacement policy.

  Tambi et al. (2006) estimated indirect costs in a sample of 12 African countries at Euro 14.7 million. Onono et al. (2014) estimated the indirect annual costs of CBPP to Kenya at USD 5.5 million per year, taking into account different control strategies used (vaccination, slaughter, treatment).

  GAPS :

  There are no studies on CBPP that measure indirect costs more precisely (see Barratt et al. 2019, Rich & Winter-Nelson, 2007; Nguyen et al., 2021 for examples in the case of other diseases).

  Greater assessment of the livelihood impacts of CBPP in a more rigorous manner is also an important gap in the literature. While the dimensions of impact on social cohesion and status have been enumerated, the use of participatory or anthropological tools to explore the nuance of these impacts is lacking, particularly in comparison to other transboundary diseases. Issues of gender dynamics that play important roles in disease control settings for poultry and small ruminants are wholly unexplored in the context of CBPP and an important area for future inquiry (see for example Bikaako et al. 2022).

• Trade implications

• Impact on international trade/exports from the EU

  Impact is high on the infected zones as trade of live cattle is not allowed.

  GAPS :

  Limited specific knowledge on actual/prospective trade bans. Mullins et al. (2000) is the closest but comprehensive analysis for FMD etc. has not been conducted.

• Impact on EU intra-community trade

  None as the disease is non-existent, but impacts would be high were an outbreak to arise in the EU.

  GAPS :

  No specific knowledge on prospective EU impacts.

• Impact on national trade

  Affected countries suffer from a ban on the exportation of live cattle. Importation of CBPP-free cattle into a CBPP affected country is also prohibited.

  GAPS :

  No specific knowledge on national trade impacts, other than analysis of Mullins et al. (2000) in Botswana.

• Links to climate

  Seasonal cycle linked to climate

  As reviewed by Provost et al. (1987), climatic conditions are more important for the way in which they affect animal husbandry than for any direct effect (e.g., animal gathering around water sources). A dry climate is believed to diminish the risk of spread, because infective aerosols from contaminated cattle dry rapidly and the pathogen is inactivated by ultraviolet rays. The rainy season may play a role in triggering infection, particularly when the animals are exposed to cool, torrential rains.

• Distribution of disease or vector linked to climate

  None.

• Outbreaks linked to extreme weather

  Outbreaks may be linked to drought, as well as floods, since both lead to congregation of cattle at water holes and higher ground respectively. Also, drought leads to long treks in search of pasture and water, which leads to stress and spread of the disease.

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

  No association has been demonstrated. However, extreme climatic conditions and socio-economic/political changes that promote mixing of infected and susceptible cattle promote disease transmission.

• Main perceived obstacles for effective prevention and control

  Diagnostics:

  There are currently severe shortages in the production and supply of cELISA and CFT kits and reagents for CBPP serology. These kits are expensive and of low sensitivity, especially for what concerns chronically-infected animals, so they must be interpreted at herd level.

  There is no data regarding the diagnostic performance (specificity and sensitivity) of commercially available PCR kits and there are no molecular assays validated under ISO17025 accreditation to guide diagnostic laboratories in the selection of routine tests for the rapid detection of the CBPP agent. The cold chain required for shipment of PCR kits induces major technical and economical constraints in Africa.

  There are no pen-side tests available for rapid CBPP diagnosis in the field.

  Farmers have no interest in diagnosing CBPP, due to punitive policies such as movement restrictions and slaughter of infected animals in the absence of compensation.

  Vaccines:

  Current vaccine production rarely meets the demand for mass vaccination campaigns of endemic African territories and the QC tools available are insufficient to guarantee their efficacy, which may vary from batch to batch and along the distribution chain.

  The stability, efficacy and duration of immunity of current live attenuated T1/44 and T1sr vaccines need to be improved. These vaccines are not thermostable and they are delivered only through trained personnel of veterinary services during vaccination campaigns. Logistical, financial and political constraints greatly limit access to vaccines (and vaccination campaigns), as well as their quality. Current vaccines provide short term immunity (less than 1 year) and at least annual revaccination is mandatory to ensure protection. Despite being transient, the humoral response elicited following vaccination is indistinguishable to that of infected animals. Occasional post-vaccinal reactions are known to occur with vaccine strain T1/44, which limit the acceptance of vaccination campaigns, weakening the efficacy of the intervention strategy.

  The current legal framework for vaccine registration may represent an issue for new generation vaccines derived from application of modern bioengineering or gene editing technologies that fall under GMO biological products.

  Production of new generation vaccines must be economically sustainable and will require investments for modernization of vaccine production plants.

  Pharmaceuticals:

  No official control strategies put in place in Africa include the use of antimicrobials. Obstacles to the integration of therapeutics are the risk of AMR development and the possible risk of creating carrier animals, though data to support the latter are lacking.

  Antimicrobials are freely available to the farmers and used with low antibiotic stewardship, which can promote the development of AMR, as increasingly reported in M. bovis strains.

  For many years, scientists have proposed to revisit therapeutics as an option, but very few reports describe studies that test the effectiveness of antimicrobials in preventing or reducing clinical signs, development of lesions, and disease transmission. The main difficulty will be regulating and monitoring antimicrobial use, while assessing its impact on the development of AMR.

  GAPS :

  Diagnostics:

  The development and validation of an alternative, highly specific cELISA test is urgently needed for use in international trade, as well as for field surveillance and prevalence studies.

  Direct molecular assays must also be developed and validated by international standards and inter-laboratory assays. Affordable freeze-dried products should be favoured to minimise cold chain requirements.

  The availability of reference standards and qualified sample panels from experimentally and naturally infected animals is very limited. These are needed for the validation of new diagnostic assays.

  Market studies are needed to assess the demand and affordability of new diagnostics and the involvement of manufacturing companies is required to target and optimise the development of inventions into successful commercial products, particularly regarding pen-side tests for rapid screening in the field.

  Vaccines:

  The current live vaccine formulation may be improved to increase their stability after reconstitution, while the use of adjuvants must be explored to improve their potency. Vaccine QC requirements must be reviewed to guarantee products with adequate efficacy. Involvement and training of veterinarians from the private sector should be explored to increase vaccine accessibility to farmers.

  Market analyses for vaccines are necessary to guide both researchers and manufacturers regarding acceptability of new products (including subunit vaccines and genetically modified strains) as well as willingness to pay (at farmer and governmental levels) and cost/benefit ratios. These studies should consider strategies for the development of multi-valent and DIVA vaccines.

  Pharmaceuticals:

  More data from in vitro and in vivo studies testing the effectiveness of therapeutics against CBPP, including field data from natural outbreaks, is necessary to allow an evidence-based decision regarding the role of antibiotic treatments in CBPP control strategies and to establish guidelines promoting antibiotic stewardship and minimising both the side effects and the development of AMR.

  The effect of antibiotic treatments on the development of immunity after recovery and in combination with vaccination must be assessed.

  Combined therapies including anti-inflammatory drugs should be explored to further reduce mortality, clinical signs and lesions.

  Vaccination alone using current commercial vaccines is not sufficient to eradicate the disease; therefore, integrated strategies, combining mass vaccination campaigns with the rational use of antibiotics and a slaughter policy, must be validated experimentally and promoted.

  Standardised protocols to determine the in vitro MIC values of Mmm strains are needed to determine epidemiological cut-off values and clinical breakpoints of Mmm to commonly used antimicrobials and to assess the acquisition of AMR by Mmm strains in the field.

• Main perceived facilitators for effective prevention and control

  Diagnostics:

  National Veterinary Services with central command, and that dispose of the required specialised manpower can greatly facilitate disease surveillance. They should dispose of well-equipped diagnostic laboratories with competent staff and working under quality assurance (ISO 17025).

  Availability of pen-side tests would allow rapid confirmation in the field (point of care diagnostics), while direct molecular diagnostics and serological assays validated by international standards would facilitate surveillance and prevalence studies.

  Capacity building on disease recognition, diagnosis, data collection and sharing, as well as reporting through increased awareness and training is needed to improve syndromic and abattoir surveillance at field level (district, region…).

  Vaccines:

  In the short term, better QC of vaccine batches is required to ensure products with adequate efficacy.

  In the mid-term, improving the stability, potency and safety of the current live attenuated vaccines would facilitate disease control, while the use of multi-valent vaccines would increase the cost-effectiveness of vaccination.

  Ultimately, the development of effective inactivated/subunit vaccines that are affordable, safe, thermostable and directly manageable by farmers would greatly facilitate the implementation of vaccination strategies.

  Furthermore, a DIVA vaccine would facilitate the implementation of combined strategies including therapeutical and/or sanitary measures, thus accelerating outbreak resolution and disease eradication.

  The development of robust and reproducible in vivo challenge models and in vitro immunoassays will be key to: i) acquire advanced knowledge on host-pathogen interactions; ii) identify the protective immune profile to be elicited with vaccination; iii) test and compare vaccine efficacy.

  Pharmaceuticals:

  Currently, CBPP is often treated unofficially by farmers with antibiotics freely available on the market. A new CBPP control strategy including the use of antimicrobials is therefore likely to meet with low barriers for implementation in the communities, as long as the recommended treatments are not too expensive. Cattle owners in CBPP endemic and surrounding areas are very aware of the disease and are willing to pay for effective measures that protect their cattle.

  GAPS :

  Diagnostics:

  Extensive comparative genomic and other “omics” analyses conducted on a large sample of strains representing the temporal and spatial diversity of Mmm and other members of the “M. mycoides” cluster are needed to determine representative pan and core genomes at different levels. These will be instrumental in (i) the development of diagnostic/genotyping tools, and (ii) the identification of the molecular basis of virulence/attenuation, host/tissue tropism and acquisition of AMR.

  A whole genome analysis approach for finest Mmm strain genotyping and comparative “omics” analyses should be developed; scripts and data repositories should be made publicly available.

  Research towards the development of sensitive kits for diagnosis at individual level is needed, as well as affordable pen-side tests.

  Vaccines:

  A clear understanding of the molecular basis of Mmm pathogenicity and protection is lacking.

  Development of more effective, safer and stable vaccines available off the shelf for trained private vets or even farmers would greatly facilitate the control of the disease.

  Pharmaceuticals:

  A policy change towards a targeted, controlled use of antibiotics may lead to improved application of effective treatment regimens as opposed to the current unofficial use. Knowledge, attitude and practice (KAP) studies should be performed in communities living in endemic regions before and during testing/implementation of new policies including antibiotics to ensure that the communication strategies are working and pitfalls leading to suboptimal use of the said antibiotics may be avoided.

Global challenges

• Antimicrobial resistance (AMR)

• Mechanism of action

  The mechanisms of AMR acquisition in Mmm are not well understood. Only a few mutations in antimicrobial target sites have been identified in relation to reduced antimicrobial susceptibility in vitro, whereas no plasmids carrying resistance genes or efflux mechanisms have been identified.

  Mechanisms leading to in vivo acquired AMR to commercially available antibiotics have been described in M. bovis and other mycoplasmas species, particularly in mycoplasmas pathogenic for humans, and are limited to target modification and ribosome protection by the tet(M) determinant (Lysnyansky and Ayling, 2016). These may be identified by molecular methods. Active efflux mechanisms have also been demonstrated in vitro. Antimicrobial resistance in both human and animal mycoplasmas have recently been reviewed by Sabine Pereyre and Florence Tardy (Pereyre, S.; Tardy, F., 2021).

  GAPS :

  The molecular mechanisms of AMR acquisition by Mmm strains must be elucidated, some of which may be identified by molecular methods based on knowledge gathered from other mycoplasma species.

• Conditions that reduce need for antimicrobials

  Implementation of mass-vaccination campaigns (targeting as much as possible the entire susceptible population) repeated at least once a year (when using T1/44), can reduce the need for antimicrobials.

• Alternatives to antimicrobials

  None, as mentioned in section 3.2, though prophylactic control by vaccination is recommended to reduce the prevalence of the disease, and test and slaughter can also be used for disease eradication.

• Impact of AMR on disease control

  No studies have so far linked directly AMR of Mmm strains and its impact on disease control.

  GAPS :

  The prevalence and impact of in vivo acquired resistance to different antimicrobials by Mmm strains is not known.

• Established links with AMR in humans

  Not studied.

  GAPS :

  The impact on human health of antibiotic treatments implemented during CBPP outbreaks needs to be evaluated, particularly in regards to the development of AMR due to poor antibiotic stewardship.

• Digital health

• Precision technologies available/needed

  No precision technologies are applied to CBPP.

  GAPS :

  Use of artificial intelligence (AI) tools for disease identification based on typical post mortem lesions could help support diagnosis in remote areas and/or when no trained personnel are available.

  Use of telemedicine, based on transferring pictures of post mortem lesions and epidemiological data on clinical cases to remote experts, may also facilitate early disease recognition and implementation of evidence-based countermeasures, resulting in reduced disease spread.

  Technologies based on applications for smartphones are likely to find a wide use, even in remote areas.

• Data requirements

  Clinical, epidemiological, pathological and laboratory data, including genomics and other “omics” data are important. Neither data collection protocols / guidelines, nor the minimum dataset requirements have been established. Data platforms for sharing and preserving information are needed.

  GAPS :

  Lack of data on disease reporting and animal movements for tracing activities and outbreak investigation represent the main challenge.

• Data availability

  Only whole and partial genome sequence data and other “omics” data, as well as extensive clinical and pathological descriptions and images are freely available through public databases and WOAH and FAO websites respectively. In addition, detailed information on the occurrence of CBPP based on official declarations is available from the WOAH information system (WAHIS).

  GAPS :

  No repositories are available for storing and updating dedicated genotyping, “omics”, antimicrobial susceptibility, epidemiological information and other relevant data related to Mmm/CBPP. Ideally, all this information should be centralised and correlated in dedicated platforms, freely accessible.

• Data standardisation

  No procedures or guidelines have been established so far.

  GAPS :

  Potential application of a model similar to the SIGMA model from EFSA for animal health data collection should be explored (EFSA 2019).

• Climate change

• Role of disease control for climate adaptation

  The role of climate change in disease control has not been studied but climate change is expected to impact animal movements and, thus, disease spread.

• Effect of disease (control) on resource use

  Limited access to land and water due to climate change are also expected to impact animal movements and transmission.

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

  None studied for CBPP, although infectious diseases are generally expected to exacerbate greenhouse gas emissions from livestock.

• Preparedness

• Syndromic surveillance

  In CBPP-free countries passive surveillance is in place based on abattoir surveillance and post-mortem investigation of animals with respiratory diseases.

  In endemic areas, the general signs associated with the disease are known by the communities and animal health workers. Syndromic surveillance has therefore played a key role in detecting the disease. However, in some cases, there are misdiagnoses (e.g., cases of pulmonary tuberculosis in cattle and Haemorrhagic septicaemia).

  GAPS :

  Constant training on disease recognition, reporting and application of early sanitary measures to all actors involved in disease control will make syndromic surveillance more effective.

• Diagnostic platforms

  In CBPP-free countries molecular diagnostics are available for the rapid confirmation of CBPP suspicions. CFT and cELISA are used for animal testing before trade.

  In endemic regions, many labs can implement ELISA techniques, which are used routinely for diagnosis, prevalence studies and international trade. CFT is also performed, although requiring skilled staff. However, Mmm isolation is rarely performed and end-point and real-time PCRs are not necessarily available; even if many labs are now equipped, skilled staff and/or reagents are often lacking. Sequencing is rarely practised because of financial limitations and logistical issues.

  GAPS :

  Sustainable and affordable point of care diagnostics (serological and high throughput assays) remain a gap in Africa.

• Mathematical modelling

  At least 13 mathematical models have been published between 2004 and 2023 in different fields. All the models include the same compartments (Susceptible, Exposed, Infectious, Recovered and Carrier states). However, the duration and the infectivity of the carrier state varies from one work to another. The estimation of R0 varies from 1. 24 in an enzootic context (Ezanno and Lesnoff, 2009) to 4.5 in an epizootic situation, using parameters from literature review (Ssematimba et al., 2015). These values imply that a herd immunity of 77% should be sufficient to interrupt transmission (Thiaucourt, 2018).

  Few models consider contact patterns among herds (Mariner et al., 2006; Ezanno and Lesnoff, 2009).

  GAPS :

  Models developed to date do not include age stratification that could be used to calibrate them. Nevertheless, the probability of becoming carriers could depend on the age of the animals.

  The role of the carrier stage in the transmission dynamics is not clear and field data are needed to validate this hypothesis.

  Furthermore, there is no clear consensus on the existence and duration of the “Recovered” state.

  Models are restricted to single herds or nearby herds and a model for geographical diffusion is still missing. A metapopulation model that takes into account movement patterns, as well as demographic dynamics, would provide hints to understand seroprevalence patterns and identify areas and periods at risk.

  Calibration on seroprevalence data by age class, together with information on mortality, would greatly improve model performances.

• Intervention platforms

  In the EU context CBPP is included in the legal framework of the recent Animal Health Law (Regulation EU 2016/429) that harmonises disease management policy from prevention to eradication. As part of the revision of the European animal health policy in 2018 (2018/1882), CBPP was classified in groups A, D and E, implying that the disease is subject to an emergency health response plan, with immediate eradication measures taken as soon as it is detected.

  In African countries there are inconsistencies across national and regional regulations and intervention strategies must be tailored based on the disease status of the territories and the capacity of veterinary services to detect and control the disease. These are mainly (i) “test and slaughter” in zones/countries that are either free of the disease or have put in place eradication programmes, and (ii) mass vaccination (using T1/44 or T1sr) in endemic regions. Cattle movement management, within individual and across neighbouring countries, is one of the most critical issues for CBPP control due to lack of platforms for animal identification and traceability. The use of integrated strategies combining the use of different control measures must be validated to promote eradication.

  GAPS :

  Harmonisation of intervention strategies and development of intervention platforms at regional (multi country) level is a priority. This will imply harmonisation of the legal framework and operational procedures for disease surveillance, diagnosis and control.

• Communication strategies

  There should be coordinated communication strategies on:

  - Importance of stakeholders in disease surveillance and control, in order to elicit changes in the behaviour of cattle owners in regards to official disease prevention and control measures. This must comprise (i) awareness and disease recognition, (ii) prevention and control measures, including benefits and drawbacks of vaccination (with anticipation of the possible advent of Willems’ reactions) and antibiotic treatments (including guidelines on antibiotic stewardship), and any compensation when test and slaughter policies are adopted.

  - Awareness of policy makers regarding the financial and logistical implications of effective control and prevention strategies, including plans for a compensation policy for cattle owners when animals are slaughtered.

  The communication strategy should be part of the intervention plan and supportive of the disease control policy adopted.

  Whenever available, communication and intervention strategies should take into consideration data on perception by farmers, both on disease and on possible intervention approaches.

  GAPS :

  Knowledge of factors influencing decision of farmers to implement disease control programmes is a key element to deliver an effective communication and intervention strategy.

Main critical gaps

• Epidemiology and impact

  • The factors influencing transmission from asymptomatic carriers to susceptible animals should be studied in more detail, either through experimental controlled settings or through follow-up field studies during an active outbreak of the disease.
  • The role of asymptomatic chronic carriers in the transmission cycle and sudden disease outbreaks should be better investigated, together with the epidemiological characteristics associated with this state (duration, proportion in the population, and transmissibility). Furthermore, it would be important to identify the conditions that favour the appearance of chronic carriers, including the role of antibiotherapy and vaccination, which mask clinical signs but do neither clear nor prevent the infection. The effect of co-infections on disease activation and outcome, by triggering outbreaks and/or persistence, must be studied.
  • Similarly, the possible role of young animals and small ruminants as reservoirs of the disease should be assessed.
  • There is limited evidence base regarding different dimensions of impact associated with CBPP, whether at macro level, impacts on trade, or specific household/livelihood impacts. Indirect and spillover effects of CBPP are particularly missing. A variety of economic models used for other transboundary diseases (notably FMD) could be used to assess CBPP impacts but have yet to be fully deployed.
  • More quantitative and qualitative assessments of the social, livelihoods, and gendered impacts of CBPP are required, over and beyond the limited case studies that exist, to develop and synthesise experiences and build a more comprehensive body of evidence in this space.

  Pathogenicity and host immune response

  • A clear understanding of the molecular basis of pathogenicity and protection is lacking, including how the host environment affects the regulation of Mmm virulence genes as well as how Mmm affects the regulation of host genes.
  • More comprehensive multi-actor in vitro models, as well as improved in vivo models, including small animal models, should be explored for the study of host-pathogen interactions and the assessment of control tools such as vaccines or treatments.
  • An in-depth characterisation of protective immune responses is needed to achieve sterile immunity.

  Control: diagnostics, vaccines and therapeutics

  • Research towards the development of more sensitive methods for individual diagnosis are needed, particularly for international trade. Affordable pen-side tests are also required.
  • In the short term, improvement of current vaccine formulations and QC procedures are necessary to increase product stability, homogeneity and potency. In the mid- and long-term new rationally-designed vaccine prototypes should be developed and tested.
  • Research should focus on improving vaccine efficacy and its duration, whilst reducing residual virulence.
  • DIVA vaccines, and their companion diagnostic test correlating serological responses to protective immunity, should also be developed, as well as multi-valent vaccines, which can greatly reduce the cost of vaccination.
  • The regulatory framework for vaccine registration should be updated for new generation engineered vaccines.
  • Data supporting or refuting the inclusion of antibiotic treatments in CBPP control strategies are needed. If supported, a policy needs to be developed including treatment regimens defining the antibiotic dosages, duration, and conditions under which they should be used in combination with other control measures (vaccinations/ slaughter/movement control etc.).
  • AMR surveillance strategies and accompanying SOPs to monitor the acquisition of resistance by Mmm strains need to be established.
  • An improved, robust challenge model is required for evaluation of novel vaccines/treatments, together with an adequate, standardised scoring system allowing a consensus methodology for the evaluation of conferred protection.

Conclusion

• Contagious bovine pleuropneumonia (CBPP) is a classic example of a transboundary animal disease requiring regional efforts and active cooperation among neighbouring countries for its prevention and control. Although its exact distribution, incidence and impact have not been well stablished, the disease is known to be endemic in sub-Saharan Africa, where it inflicts a severe socio-economic impact by affecting peoples’ livelihoods and wellbeing. Test and slaughter, animal movement control and mass vaccination are the main strategies currently recommended and partly applied for disease control. However, their effectiveness is heavily diminished, since testing is expensive and not always available, no compensation is given to affected cattle owners (a clear determent to test and report the disease), and animal identification and movement control systems are not functional in most affected countries. Furthermore, existing diagnostics are for interpretation at herd level only and DIVA tests are not available. Live attenuated vaccines (T1/44 and T1sr) are available for controlling the disease but their use strictly depends on the financial resources and quality of the national veterinary services that are responsible for their implementation. Vaccine safety, efficacy, stability and availability issues further hamper the effectiveness of vaccination as a stand-alone tool for disease control and it is clear that mass vaccination alone is not sufficient to eradicate the disease. Therefore, new combinations of implementable intervention strategies are necessary, which should include the rational and controlled use of antibiotic treatments, coupled to antimicrobial susceptibility testing for the surveillance of AMR emergence in the field.

  As a transboundary disease, where animal mobility plays a pivotal role in the rapid geographical spread at national and international levels, efforts should be made towards the collection, centralisation and harmonisation of i) livestock mobility, ii) disease prevalence, and iii) socioeconomic impact data at different temporal and spatial scales to identify areas at risk and support the implementation of appropriate control strategies. All these data should be incorporated in mathematical models to better assess the impact of new combinations of control measures.

  Availability of diagnostics is currently an issue. With new policies governing the implementation of CBPP control strategies, there may be an increased demand for diagnostics (serological and direct detection, lab-based and pen-side, DIVA). Market studies to identify financially sustainable diagnostics, bearing this development in mind, would be most helpful.

  Knowledge in disease pathogenesis and protective immune responses has been limited due to the absence of in vivo and in vitro reliable infection models and, until recently, the lack of bioengineering tools such as CRISPR/Cas9 base editor systems functional in mycoplasmas. Research efforts should focus on application of omics technologies and reverse vaccinology for a better characterization of the host-pathogen interplay, the identification of correlates of protection and the development of rationally-designed vaccines. The development of robust in vivo and in vitro challenge models remains essential to speed up vaccine development and validation. In the meantime, the regulatory framework for vaccine registration in the region must be updated to include new generation, engineered vaccines. Also, while awaiting novel, improved diagnostics and vaccines, implementation of the currently available ones must be optimised. Quality control during and after manufacturing of these tools must also be improved. Finally, training of personnel on disease recognition and awareness remains a fundamental and cost-effective tool to support disease control and should be further expanded using modern technologies such as distance learning, telediagnosis, and artificial intelligence tools, coupled to smart applications for disease reporting.

  In conclusion, improved vaccines and diagnostics are needed to assist in CBPP control and eradication strategies, together with implementation of control policies. To aid the development of these new tools and policies, a better understanding of the pathogen and its interaction with the host is needed. Also, other factors (social, political, financial, biological, etc), involved in the persistence and spread of the disease, must be investigated. With sufficient financial, scientific and political support, CBPP can be eradicated.

Sources of information

• Expert group composition

  Lucia Manso-Silvan, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), France – [Leader].

  Andrea Apolloni, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), France.

  Elise Schieck, International Livestock Research Institute (ILRI), Kenya.

  Flavio Sacchini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Italy.

  Geoffrey Munkombwe Muuka, Central Veterinary Research Institute (CVRI), Zambia.

  Karl M. Rich, Oklahoma State University, USA.

  Lamya Rafi, MCI Santé Animale, Morocco.

  Loic Comtet, Innovative Diagnostics, France.

  Martin Heller, Friedrich Loeffler Institut (FLI), Germany.

  William Amanfu, independent consultant, Ghana.

• Date of submission by expert group

  10^th of July 2023.

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