Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia.

Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine.

Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine.

Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides.

The current control method for contagious bovine pleuropneumonia (CBPP) in Africa is vaccination with a live, attenuated strain of Mycoplasma mycoides subsp. mycoides (Mmm). However, this method is not very efficient and often causes serious adverse reactions. Several studies have attempted to induce protection using inactivated mycoplasma, but with widely contradictory results. Therefore, we compared the protective capacity of the live T1/44 vaccine with two inactivated preparations of Mmm strain Afadé, inoculated with an adjuvant. Protection was measured after a challenge with Afadé. The protection levels were 31%, 80.8% and 74.1% for the formalin-inactivated, heat-inactivated and live attenuated preparations, respectively. These findings indicate that low doses of heat-inactivated Mmm can offer protection to a level similar to the current live attenuated (T1/44) vaccine formulation.

Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA.

BACKGROUND: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the "mycoides cluster" frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. RESULTS: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the "mycoides cluster". Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. CONCLUSIONS: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Efficacy of two vaccine formulations against contagious bovine pleuropneumonia (CBPP) in Kenyan indigenous cattle.

A live, attenuated vaccine is currently the only viable option to control of CBPP in Africa. It has been suggested that simple modifications to current vaccines and protocols might improve efficacy in the field. In this report we compared the current vaccine formulation with a buffered preparation that maintains Mycoplasma viability at ambient temperature for a longer time. Groups of animals were vaccinated with the two formulations and compared with non vaccinated groups. Half of the animals in each group were challenged 3 months post vaccination, the other half after 16 months. Protection levels were measured using the pathology index, calculated from post mortem scores of lesions from animals killed during the course of clinical disease. In the challenge at 3 months post vaccination, the protection levels were 52% and 77% for the modified and current vaccine preparations, respectively. At 16 months post vaccination, the protection levels were 56% and 62% for the modified and current vaccine preparations, respectively. These findings indicate that there are no differences in protection levels between the two vaccines. Because of its longer half life after reconstitution, the modified vaccine might be preferred in field situations where the reconstituted vaccine is likely not to be administered immediately.

Assessing the effectiveness of intubation as a challenge model in contagious bovine pleuropneumonia vaccine experiments.

A study was carried out to assess the effectiveness of a bronchoscope in administering a pathogenic field strain of Mycoplasma mycoides subsp. mycoides (MmmSC) in cattle challenge experiments. Out of 16 animals inoculated using the bronchoscope, 10 (62.2%) showed clinical disease as evidenced by fever and 15 (93.8%) displayed typical lesions of CBPP from which MmmSC was isolated. Serum samples collected weekly were tested by Complement Fixation Test (CFT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibodies to MmmSC were detected in 10 out of the 16 animals by the CFT and 11 out of the 16 animals by c-ELISA. The onset of clinical disease was as early as 2 days post-inoculation, and most of the animals developed clinical disease 2 to 3 weeks post-infection. These results clearly demonstrate that nasotracheal inoculation of pathogenic strain of MmmSC with the aid of a bronchoscope can lead to early onset of clinical disease; similar to previous studies but with higher numbers of animals showing clinical disease. This is in contrast with previous studies where early clinical disease was observed in as little as 15% of inoculated animals. This nasotracheal inoculation method using a bronchoscope can, therefore, be adopted for use in experimental challenge infections of cattle. This method is found to be a better replacement to the contact transmission method whose drawback includes extra cost of donor animals and unpredictable rate and timing of transmission from intubated to challenge animals.

Assessment of in vitro interferon-gamma responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type.

Contagious bovine pleuropneumonia (CBPP) is a lung disease caused by the bacterial pathogen Mycoplasma mycoides ssp. mycoides small colony type (MmmSC). It has been spreading due to a number of factors including poor vaccine efficacy and poor sensitivity of current diagnostic tests. The purpose of this study was to assess interferon gamma (IFN-gamma) release after stimulation of peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. PBMC collected from 15 artificially infected animals were incubated with different concentrations of total MmmSC antigen. After 72h of incubation the IFN-gamma release was measured and found to be elevated in 11 animals. We did not observe a correlation between IFN-gamma release of animals with and without pathomorphological gross lesions. Therefore, our data do not confirm a role for CD4 T-lymphocytes in protection, since there is no correlation between IFN-g secretion (supposed to be mainly derived from CD4 T-cells) and disease severity. Additionally, we applied immunocytochemistry on affected lung tissue and detected no build up of T-lymphocytes (CD4 T-cells, CD8 T-cells) but a high presence of myeloid cells.