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Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

A better understanding of protective immune memory against contagious bovine pleuropneumonia (CBPP) is needed in order to facilitate the development of safer vaccines based on selected components of the pathogen. For this purpose, cells collected from lymph nodes draining the lungs of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC)-infected cattle were stimulated with the pathogen in vitro and evaluated concurrently for proliferation (CFSE based method), expression of activation, memory markers and cytokine production. Direct evidence is presented for a major contribution of CD4+ T cells to the vigorous proliferative and T1 biased cytokine recall responses observed in cattle that have recovered from infection but not in animals developing the acute form of the disease. Two different phenotypes of MmmSC-specific memory CD4 were observed based on CD62L expression and proliferative capacities. Furthermore, recall proliferation of B cells also occurred but was strictly dependent on the presence of CD4. The information provided in this study will facilitate the search for MmmSC antigens that have potential for the development of subunit vaccines against CBPP.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), is still a major cattle disease in Africa. Development of long-term protective vaccines, the only relevant strategy to achieve CBPP eradication, requires the characterisation of the protective immune mechanism. To this aim, the present study investigated the cellular immune response persisting in the lymph nodes of cattle infected naturally and experimentally by contact, one year post exposure. The lymph node cell composition, MmmSC responsiveness and phenotype of the MmmSC-responding lymphocytes were compared between animals according to the different outcomes of the infection. To unravel the protective mechanism, the study focussed on the MmmSC-specific memory immune response generated in recovered cattle, known to develop long-term immunity and to be resistant to reinfection. An MmmSC-specific immune response, mediated by IFNgamma-secreting CD4 T-cells, was detected in the lymph nodes of all recovered cattle. Furthermore, the magnitude of this immune response was significantly higher in animals with complete recovery than in recovered animals presenting lung sequestra. The findings suggest that, in recovered cattle, a subset of MmmSC-primed IFNgamma-secreting CD4 T-cells homed to the regional lymph nodes as MmmSC-specific memory T-cells, likely responsible for the protective anamnestic response. Induction and expansion of this subset of MmmSC-specific CD4 memory T-cells might be a major goal to develop efficient long term protective vaccines against CBPP.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most significant cattle disease in Africa. The control measures, which led to eradication from numerous countries are not feasible in Africa where the only prophylaxis relies on vaccination. However, the attenuated vaccines, used up to now in Africa, are of low efficiency. The development of an improved vaccine is, therefore, a necessity. The purpose of this study was to compare some immunological parameters in MmmSC-infected cattle (endobronchial versus natural in-contact infection) and assess the response in correlation with the clinical outcome (death versus recovery). Characterization of the immune parameters elicited in recovered animals, known to be refractory to new infection, will be an important step towards development of new vaccines against CBPP. A significant outcome of this study was the demonstration that all MmmSC-infected cattle developed a MmmSC-specific cell-mediated immune response. A kinetic analysis of the MmmSC responsiveness showed that the main difference between endobronchially- and in-contact infected animals was the delay before the onset of the MmmSC-specific immune response. The first MmmSC-responding PBMC sample was selected from each animal for cell phenotyping. The phenotypic analysis of this early MmmSC-induced response revealed the predominant contribution of the CD4 T-cells in all animals whereas IFNgamma was only constantly produced in recovered animals. Evolution of this early MmmSC-specific immune response was then followed by a kinetic analysis of the MmmSC-induced CD4 T-cell response and IFNgamma released. The results demonstrated that in recovered animals, the MmmSC-specific CD4 Th1-like T-cell response was maintained until slaughtering whereas in animals with acute disease, progression of CBPP was associated with a decreased ability of the PBMC to produce IFNgamma. The results led to the identification of immune parameters, which correlate with protection against CBPP and to a relevant strategy for the development of improved vaccines against this disease.

Contagious bovine pleuropneumonia (CBPP) is a contagious infection of cattle caused by a mycoplasma, M. mycoides subsp. mycoides SC (MmmSC). It induces lesions of pleuropneumonia in acute cases and the formation of pulmonary "sequestra" in chronic cases. The disease is prevalent mostly in Africa, where it is responsible for high losses, but it has also been sporadically present in Southern Europe until 1999. Vaccination is now prohibited in most countries except in Africa. An empirical "inoculation" procedure was developed as early as 1852 in Europe but it may have been used even earlier in Africa. The inoculation of pleural fluid was performed at the tip of the tail in Europe and on the bridge of the nose in Africa. It conferred good protection but induced a high number of fatal cases. Various inactivated preparations have been tested in the past with inconclusive results leading sometime to some protection and some other time to a sensitisation of the immunised animals. Such preparations have never been used in the field. Attenuated MmmSC strains have been developed in the 1950s and used extensively in the field both in Africa and Australia. The best known vaccine strains are KH3J, T1/44 and T1sr. Vaccination campaigns have succeeded in reducing considerably the CBPP prevalence in these two continents but eradication was achieved in Australia only by switching to strict measures of animal movement control and a stamping-out policy. The search for new CBPP vaccines has become a major issue for African countries that are facing an increase in outbreaks. The rationale for this search is based on a better understanding of the mycoplasma virulence mechanisms that could lead to a targeted attenuation of MmmSC strains. It is also based on a better understanding of the bovine immune response that may be driven to a pathogenic inflammatory response or conversely to a better balanced response leading to protection.

The control of contagious bovine pleuropneumonia (CBPP) has been clearly identified by the Organisation of African Unity/Inter-African Bureau of Animal Resources as a priority. In the first part of this article, the authors introduce the past and present vaccines, based on the two classic strains, T1, and KH3J. They describe the guidelines for vaccine production technology, and the quality control requirements for CBPP vaccines of the Office International des Epizooties. The failure of the currently used T1-SR vaccine to provoke satisfactory immunity in cattle, particularly in the newly infected areas of Africa, is pointed out. Other shortcomings of the current CBPP vaccines are also highlighted. Thus, there is a need to improve CBPP vaccines and the authors propose detailed emergency measures to address this problem. In the second part of the article, a subunit approach using immunostimulating complex technology is outlined. The authors emphasise the importance of current research in cell-mediated immunity and immunopathology, which is aimed at improving the efficacy of CBPP vaccines.

While it is easy to diagnose contagious bovine pleuropneumonia (CBPP) in an animal in the acute clinical stage, subacute and chronic forms are more difficult to diagnose. Recourse to laboratory tests is essential to confirm any suspicion of CBPP. As standard diagnostic procedures (isolation, culture, biochemical tests, serological tests) are lacking in specificity and sensitivity, improvements are needed. Progress in molecular biology techniques has led to new tests, among which are the polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Application of these techniques to CBPP offers a number of advantages, and has considerably enhanced the specificity and sensitivity of diagnosis.

A new selective assay for the detection of Mycoplasma mycoides subsp. mycoides SC (MmmSC) via the polymerase chain reaction (PCR) has been developed. This test used two PCR assays: a control-PCR (MYC-PCR) identifying the pathogen as a member of the mycoides cluster and the MSC-PCR which is specific for MmmSC. The MYC primers targeted a DNA sequence of about 460 bp from all the 59 mycoides cluster-strains tested. No amplification occurred with bovine genomic DNA or with the 11 other bacterial species assayed. The MSC primers selectively amplified a 275 bp sequence from the 27 MmmSC strains tested, with three specific internal restriction sites allowing confirmation of the identification. The sensitivity assessed by direct agarose gel analysis for both PCR assays was 100 CFU. The sensitivity of the MSC-PCR was increased to 1 CFU by a dot-blot hybridization step using, as a probe, the entire 275 bp sequence digoxigenin-labeled by PCR. These two PCR assays were successfully used to detect MmmSC in pleural fluids from naturally-infected cattle. We conclude that these two PCR assays may be valuable tools for the diagnosis of contagious bovine pleuropneumonia.