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Contagious bovine pleuropneumonia (CBPP) is a bacterial disease caused by Mycoplasma mycoides subsp. Mycoides. This disease affects ruminants mainly cattle with respiratory disorders as predominant symptoms. In Burkina Faso, this condition has been considered as enzootic since several years but data on its seroprevalence remains scares. This study aimed to establish the serological prevalence and determinants of CBPP in Burkina Faso in 2017. For this purpose, 3969 serum samples have been collected following a stratified sampling plan based on vaccination coverage in 12 regions, 84 communes, and 210 villages and analyzed using c-ELISA test. Individual seroprevalence was 16.91% (95% CI: 15.74-18.07%), while 84.5% (95% CI: 60.46-80.02%) of communes, chosen as epidemiological units were found positive. The individual prevalence was found to be associated with agro-ecological area (p < 0.05) and a prevalence of 18.70% (95% CI: 16.74-20.66%) was noted in Sahelian areas, while 15.79% (95% CI: 14.34-17.23%) was found in Soudanian areas. The prevalence was also significantly associated with vaccination coverage (p < 0.05) with a prevalence of 13.92% (95% CI: 11.66-16.18%), 19.21% (95% CI: 16.66-20.75%) and 11.61%(95% CI: 9.00-14.23%) for high, moderate, and low vaccination coverage respectively. The individual prevalence was respectively 16.97 (95% CI: 15.56-18.39%) and 17.13% (95% CI: 15.93-18.33%) for female and animals more than 2 years old. According to regions, the highest seroprevalence was found in Plateau Central region (38.18%, 95% CI: 29.1-47.26%), while the lowest was found in Centre-Est Region (7%, 95% CI: 4.5-9.5%). These prevalence data will allow us to adapt the ongoing strategy to control CBPP in Burkina Faso.

Contagious bovine pleuropneumonia (CBPP) was recognised on Bako Agricultural Research Farm, in the Oromia Region of Ethiopia, for the first time on 5 May 2011. The outbreak was investigated by combining recognition of clinical signs, post-mortem examination, mycoplasma isolation and serological testing using competitive enzymelinked immunosorbent assay (c-ELISA). The clinical cases were monitored for eight months; sick animals were treated with a range of antibiotics and isolated if necessary. The outbreak of CBPP was confirmed both bacteriologically and serologically and had spread to almost the entire herd (96.7%) within the eight-month observation period. Of the animals that recovered after antibiotic treatment, 12.3% fell sick again, showed typical signs of CBPP and were considered to be carriers. The role of treatment in the prevention of the spread of CBPP was minimal. Newly purchased animals that were not tested and quarantined before being introduced onto the farm were suspected to have been the most probable source of infection., (© OIE (World Organisation for Animal Health), 2016.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC&#95;0136 and MSC&#95;0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease, is characterized by massive inflammation of the lung especially during the acute clinical stage of infection. Tissue samples from cattle, experimentally infected with Mycoplasma mycoides subsp. mycoides Afadé, were subjected to histopathological and immunohistochemical examination in order to provide insight into innate immune pathways that shape inflammatory host responses. Lung lesions were characterized by vasculitis, necrosis, and increased presence of macrophages and neutrophils, relative to uninfected animals. The presence of three cytokines associated with innate inflammatory immune responses, namely, IL-1β, IL-17A, and TNF-α, were qualitatively investigated in situ. Higher cytokine levels were detected in lung tissue samples from CBPP-affected cattle compared to samples derived from an uninfected control group. We therefore conclude that the cytokines TNF-α and IL-1β, which are prevalent in the acute phase of infections, play a role in the inflammatory response seen in the lung tissue in CBPP. IL-17A gets released by activated macrophages and attracts granulocytes that modulate the acute phase of the CBPP lesions.

Mycoplasma mycoides subsp. mycoides is the causative agent of contagious bovine pleuropneumonia. A pathogenic strain BEN-1 was isolated from bovine lung and underwent continuous passages in rabbits for 468 generations. During this process, the strain's strong virulence became weak and, gradually, it lost the ability to confer protective immunity in cattle but developed virulence in rabbits. In order to gain insight into the mechanisms behind the reduction in virulence and the loss of immunogenicity, we sequenced five representative strains of the BEN series, including the original strain (BEN-1), the strain generation that first acquired virulence in rabbits (BEN-50), the two vaccine strain generations (BEN-181 and BEN-326), and the strain generation showing the greatest loss of immunogenicity (BEN-468). The gene mutation rate in the four different propagation stages varied greatly, and over half of variations observed in each generation were removed during the propagation process. However, the variation maintained in the BEN-468 generation might contribute to its changes in virulence and immunogenicity. We thus identified 18 genes associated with host adaptation, six genes contributing to virulence in cattle, and 35 genes participating in conferring immunity in cattle. These findings might help us optimize the vaccine to obtain more effective immunization results.

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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Contagious bovine pleuropneumonia (CBPP), an infection of cattle caused by the small colony biotype of Mycoplasma mycoides subspecies mycoides (MmmSC), is a significant constraint to improved pastoral cattle productivity in sub-Saharan Africa. This cross-sectional study was aimed to estimate serological prevalence of CBPP and identify risk factors for herd sero-positivity within agro-pastoral areas of Nigeria., Results: The herd level prevalence of CBPP was 54.7% (95% confidence interval (CI) = 47.7-62.0), and proportion of animals with detectable MmmSC monoclonal antibody was 30.2% (95% CI = 26.3-34.4). Herds were more likely to be sero-positive if they were potentially exposed to recent CBPP outbreaks (odds ratio (OR) = 4.9, 95% CI = 2.4-10.1) or of larger sizes (OR = 3.0, 95% CI = 1.2-7.5). Herds vaccinated against the disease had lower odds of being sero-positive (OR = 0.12, 95% CI = 0.02-0.6) than unvaccinated herds., Conclusions: CBPP is endemic to agro-pastoral areas, and it is doubtful if the current control strategies are making real impact in reducing production losses. Although eradication is more likely to be achieved through regional approaches, enhanced vaccination coverage supported with targeted surveillance and a trace back system based on cattle trade and movement records will sustain effective control of the disease in the Nigerian cattle population.

Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP., (Copyright © 2015 Mulongo et al.)

A serological survey to determine the prevalence of contagious bovine pleuropneumonia (CBPP) in Mali was carried out by using the competitive enzyme linked-immunosorbent test (c-ELISA) on 8007 serum samples systematically collected from 199 cattle herds collected throughout the whole country. Results showed a national prevalence of 18.11 % at the individual level and 85.93 % at the herd level. Significant variations in the individual prevalence were observed between regions of the country and ranged from 4.63 % in Tombouctou to 54.88 % in Kidal. At the herd level, although there were variations between regions, a high prevalence was constantly observed ranging from 60 to 100 %, hence confirming the endemic nature of the disease across the country. The CBPP risk varied also between regions and was very low in Tombouctou (odds ratio (OR) = 0.4) but very high in Kidal (OR = 9.8). Similarly, the risk seemed higher in the animals of the over 3-year age group (OR = 1.6) compared to the other age groups. It was also observed that there was a slightly higher risk (OR = 1.3) in the females than in the males. This study confirms the presence of CBPP across the country and should help to elaborate strategies for the effective control of the disease.

Mycoplasma mycoides subsp. mycoides (Mmm) strain Afadé had previously been shown to undergo spontaneous phase variations between an opaque capsulated variant and a translucent (TR) variant devoid of a capsule but able to secrete cell-free exopolysaccharides. This phase variation is associated with an ON/OFF genetic switch in a glucose permease gene. In this study, in vivo and in vitro assays were conducted to compare the virulence of the two variants and their abilities to resist host defence. Capsulated variants were shown, in a mouse model, to induce longer bacteraemia that was correlated with better serum resistance in vitro. In contrast, TR variants displayed better ability to adhere to an inert support, linked to the absence of a capsule, changes in cell surface hydrophobicity and increased resistance to antimicrobial peptide and hydrogen peroxide. The switch from one variant population to another, which was observed both in vivo and in vitro under stress conditions, is further discussed as a means for Mmm to modulate its interactions with animal hosts during different stages of the disease., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm) is endemic in many African countries due to fragmented veterinary services and the lack of an efficient vaccine and sensitive diagnostics. More efficient tools to control the disease are needed, but to develop the tools, a better understanding of host-pathogen interactions is necessary. The aim of this study was to characterize the kinetics of the humoral immune response against 65 Mmm surface antigens for an extended period in cattle that survived a primary infection with Mmm. We describe clinical and haematological outcomes, and dissect the humoral immune response over time, to specific antigens and compared the antibody responses between different pathomorphological outcomes. No antigen-specific antibodies correlating with protection were identified. Interestingly we found that animals that developed Mycoplasma-containing sequestra had significantly higher antibody levels against proteins comprising the surface proteome than the animals that cleared Mycoplasma from their lungs. Based on these data we suggest that high antibody titres might play a role in the establishment of pathomorphological changes, such as vasculitis, which should be investigated in future studies. Beneficial antibody specificities and cellular immune responses need to be identified in order to foster the development of an improved vaccine in the future., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides (Mmm) is an economically very important cattle disease in sub-Saharan Africa. CBPP impacts animal health and poverty of livestock-dependent people through decreased animal productivity, reduced food supply, and the cost of control measures. CBPP is a barrier to trade in many African countries and this reduces the value of livestock and the income of many value chain stakeholders. The presence of CBPP also poses a constant threat to CBPP-free countries and creates costs in terms of the measures necessary to ensure the exclusion of disease. This opinion focuses on the biomedical research needed to foster the development of better control measures for CBPP. We suggest that different vaccine development approaches are followed in parallel. Basic immunology studies and systematic OMICs studies will be necessary in order to identify the protective arms of immunity and to shed more light on the pathogenicity mechanisms in CBPP. Moreover a robust challenge model and a close collaboration with African research units will be crucial to foster and implement a new vaccine for the progressive control of this cattle plague.

The authors have described an epizootic infection of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), that has affected Ndama bovine in Lounthy village, a locality based in Bala city in the Eastern part of Senegal, during the post-rainy season in November 2012. After the cessation of vaccination, a hotbed of suspicion of CBPP was identified on November 3rd 2012 in the village of Lounthy: out of the total of 98 cattle, 13 animals were sick and 5 of them died. These studies have been done according to clinical aspects, serological, bacteriological and molecular analysis of the samples. This reemergent disease will give new orientations for CBPP control in Senegal, where it was supposed the disease has been eradicated since 2005.

A cross sectional study was undertaken from October 2010 to March 2011 to determine the seroprevalence of contagious bovine pleuropneumonia (CBPP) and its related risk factors in export quarantine centers. A total of 3,111 cattle sera were collected from different export quarantine farms located in and around Adama, namely, Bekero, Jogo, Kedir, and Dera farms, and tested for the presence of Mycoplasma mycoides subsp. mycoides small colonies antibody using competitive enzyme-linked immunosorbant assay. Of the total 3,111 cattle sera examined, 124 (4 %) were found positive for CBPP. Among the potential predisposing factors assessed, origin, transportation condition, confinement level, and stay time of the animals in quarantine center were not found significantly (P > 0.05) associated with the occurrence of the disease. Whereas age was found significantly (P < 0.05) associated with the occurrence of the disease in which a high seroprevalence was recorded in aged (9.5 %) animals than young (3 %). Generally, this study showed that CBPP is a threat for Ethiopian livestock export market and a well established disease in Borana and Bale areas, where the animals originated.

Mycoplasma bovis and Mycoplasma mycoides subspecies mycoides small colony (MmmSC) are causes of bovine mycoplasmosis and contagious bovine pleuropneumonia (CBPP), respectively, and are responsible for serious economic losses in cattle around the world. CBPP was last reported in Poland in 1939 but bovine mycoplasmosis is believed to be endemic. A survey of 3670 serum samples for antibodies to M bovis and MmmSC from 361 herds in 16 Polish provinces Poland between 2007 and 2010 found no evidence of CBPP. The seroprevalence of M bovis, however, appeared high with 76.7 per cent of samples giving a positive reaction in the ELISA test, which did not appear to reflect the clinical disease status of the cattle. Adjusting the sensitivity of the test reduced the prevalence to 28.2 per cent and reflects the levels reported in other European countries.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type, was once the most damaging infectious animal disease in China, second only to rinderpest. Between 1949 and 1989, 178,570 cattle died of CBPP, causing estimated losses of 356 million RMB (1RMB=approx. £0.094, US$0.15, €0.11 at 27th January 2011). In 1956, in an effort to control the disease, a virulent strain of the causative organism (Ben-1) was attenuated by multiple passages in rabbits. The resultant vaccine achieved a high protection rate in cattle with a duration of immunity of 28 months. Vaccines were also prepared in sheep to increase the antigen yield and then in Tibetan sheep because it caused fewer adverse reactions in yaks and related species. The last CBPP infected animal was identified in 1989 since when no more cases have occurred. In 1992, vaccination of cattle was stopped. In 2008 China submitted an application to the World Organization for Animal Health (OIE) to be declared CBPP-free., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.

The complement fixation test (CFT), the c-ELISA and an indirect LppQ ELISA were compared to post-mortem (PM) inspection for the diagnosis of contagious bovine pleuropneumonia (CBPP). Sera from 797 cattle in the CBPP affected area of Kazungula, Zambia and 202 sera from Lusaka, Zambia, a CBPP-free area were used. The clinical history of CBPP was recorded and all the cattle from Kazungula were slaughtered and PM inspections conducted. The prevalence of CBPP in Kazungula was 67.5% (95%CI 67.2%, 70.8%), 52.6% (95%CI 49.2%, 56.2%), 59.0% (95%CI 55.5%, 62.4%) and 44.4% (95%CI 41.0%, 47.9%) using PM inspection, CFT, c-ELISA and LppQ ELISA, respectively. Three of the 202 negative control animals tested positive on the c-ELISA although they were from a known CBPP negative zone. In this study, the c-ELISA was more sensitive in detecting cattle with lesions in the chronic stage than any other test whilst the CFT detected more during the onset stage. No single serological test could detect all stages of CBPP infection, therefore the use of more than one test is advised.

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.

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.

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology. We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross-reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC&#95;0636), a glycosyl transferase (MSC&#95;0108), and an acyl carrier protein phosphodiesterase (MSC&#95;0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in Escherichia coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100% diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70% and 60%, respectively., (Copyright 2009 Elsevier B.V. All rights reserved.)

Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead-based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to those of the untreated controls. In correlating protein-specific humoral responses to T1/44-induced immunity, five proteins associated with a protective immune response were identified by statistical evaluation, namely, MSC&#95;1046 (LppQ), MSC&#95;0271, MSC&#95;0136, MSC&#95;0079, and MSC&#95;0431. These five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10(-6). Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests. In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

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.

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

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 var. mycoides small colony (MmmSC), is one of the most important diseases of cattle in Africa. The role of innate or acquired cell mediated and humoral immunity in conferring protection against MmmSC infection has not yet been elucidated. On the other hand, the pathological lesions caused by the aetiological agent have been considered indicative of an immunopathological process. In this study ten naïve cattle were exposed to in-contact infection with animals infected by intubation with a strain of MmmSC. Clinical signs, antibody response, IFNgamma release and pathological changes at necropsy were analysed and compared with the events following in-contact infection of an equal number of animals kept under daily treatment with cyclosporine for the entire observation period of 84 days. Cyclosporine is a suppressor of the immune response related to the T-cell system. Under the conditions of the experiment, cyclosporine appeared to condition the pathogenesis of CBPP by delaying the events that follow infection, bringing further support to the possibility that the immune response may have an impact on the disease outcome.

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.

The gene sequence coding the N-terminal domain of LppQ was amplified from Mycoplasma mycoides subsp. mycoides SC (MmmSC) HVRI X strain by PCR using special primers and was cloned into the EcoR I /Sal I sites of pET32a vector to construct the expression recombinant plasmids. The recombinant plasmids were indentified by restriction digestion, PCR and sequence analysis. The gene was overexpressed in Escherichia coli BL21 (DE3) host cell and the soluble protein was purified with Ni-NTA His. Bind purification kits. The amount of recombinant protein reached 53.7% of the total mass of bacterial protein. The purity of recombinant protein reached to over 95 %. The antigen activity of the purified protein was examined with Western blot analysis. The purified protein reacted strongly with the standard positive serum and didn't react with the negative sera of contagious bovine pleuropneumonia(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 major threat for cattle health and production in Africa. This disease is caused by the small-colony type of Mycoplasma mycoides subspecies mycoides (MmmSC). Transmission occurs from direct and repeated contacts between sick and healthy animals. Veterinary services recently reported a resurgence of CBPP in the province of West Wellega, in the Ethiopian highlands. A research program was set up to estimate the epidemiological parameters of the within-herd infection spread. A follow-up survey was implemented in 71 sampled herds of the Boji district (West Wellega province). Fifteen herds were classified as newly infected and used in a serological- and clinical-incidence study. The overall 16-month cumulative sero-incidence risk was 34%. Clinical cases were recorded for 39% of the seropositive cattle; case-fatality risk was 13%. There was no evidence of benefit on infection spread of CBPP-control measures used locally by farmers (isolation or antibiotic treatments of sick animals). This might be related to a lack of power in the statistical analyses or to a quality problem for the medications used (and more generally, for health-care delivery in the Boji district).

A study was carried out on four adult cattle to assess the pathogenicity of Mycoplasma mycoides subsp. mycoides SC strain T1/44, currently used as a vaccine for the control of contagious bovine pleuropneumonia (CBPP) in Namibia. Post mortem examination 9 weeks after endobronchial inoculation of the vaccine strain to three of the four animals revealed unilateral pleuropneumonic lesions, pleuritis and well-developed sequesters in two of the three inoculated animals and several small sequesters surrounded by pleuropneumonic lesions in the diaphragmatic and apical lobes in one animal. The fourth animal, which was not directly inoculated but was in close contact with the inoculated animals, revealed only an adhesion area of the lung to the ribcage. Serological examination carried out using the complement fixation test (CFT) detected positive titres in all three intubated animals and the indirect CBPP-LppQ-ELISA was positive for two of the three inoculated animals. The contact animal showed no seroconversion. M. mycoides subsp. mycoides SC was isolated from the sequesters of two of the inoculated animals. Isolation of mycoplasmas was not possible from the third inoculated animal due to heavy contamination of the samples by other bacteria, but the presence of M. mycoides subsp. mycoides SC could be evidenced by PCR from clinical samples. The identity of the T1/44 vaccine strain isolated from the sequesters of two animals was confirmed by T1/44-specific PCR analysis and by IS1296 typing using Southern blot. These results clearly show that inoculation of T1/44 vaccine via the endobronchial route can lead to CBPP.

The experimental field trial with an immunostimulating complex (ISCOM) vaccine has been an occasion to explore the role of a Th1 response in the pathogenesis caused by Mycoplasma mycoides subsp. mycoides small colony (MmmSC) and in immune protection. The ISCOM complex is known to promote Th1 response. Antibodies to MmmSC were detected by indirect enzyme-linked immunosorbent assay (ELISA) in the vaccinated cattle, although the levels were lower than in a previous study. No antibodies were detected by complement fixation test (CF). After the challenge infection, vaccinated animals developed CF antibody response. They showed significantly reduced mortality compared with controls. However, gross pathological and histopathological score for vaccinated animals was as high as for the non-vaccinated, characterized by a high inflammatory reaction with histopathology dominated by interlobular pneumonia with vasculitis.

Four serological tests were compared in order to evaluate their efficacies in detecting antibodies to M. mycoides subspecies mycoides SC in cattle sera sampled in 1995 from herds affected with contagious bovine pleuropneumonia (CBPP) in the north-western part of Botswana. Tests that were compared included immunoblotting test (IBT), indirect enzyme-linked immunosorbent assay (i-ELISA), competitive enzyme-linked immunosorbent assay (c-ELISA) and complement fixation test (CFT). The percentages of seropositive samples in the iELISA (48%) and in the c-ELISA (48%) were similar but were comparatively lower than those obtained by the IBT (57%) and CFT (61%). The percentages of positive sera in the IBT and CFT were also similar and overall the efficacy of these tests was better than that of the two ELISA tests. There was 95.5% agreement between the IBT and CFT, 85% agreement between the IBT and c-ELISA, 90.9% agreement between the IBT and i-ELISA, 88.6% agreement between the i-ELISA and CFT, 80% agreement between the c-ELISA and CFT and 90% agreement between the two ELISA tests. It became clearly evident from this comparative study that no single serological test was capable of detecting all animals affected by CBPP under natural field conditions of infection.

Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.

The immunogenicity of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) vaccines was investigated in BALB/c mice. Groups of mice were vaccinated with either (1) unconjugated capsular polysaccharide (CPS), (2) CPS covalently conjugated to ovalbumin via a carbodiimide reaction, (3) CPS non-covalently bound to latex microspheres, (4) CPS non-covalently complexed with rabbit anti-CPS IgG, and (5) whole inactivated, ultrasonically disrupted (WID) MmmSC. Only mice immunized with the CPS-ovalbumin conjugate exhibited a significant (P<0 small middle dot001) antibody response against CPS. Mice immunized with WID vaccine exhibited a high ELISA antibody titre against non-CPS (protein) antigens only. Mice given WID vaccine were immune against challenge with live MmmSC, and exhibited a significantly reduced degree of mycoplasmaemia (both in incidence and duration) as compared with non-vaccinated controls (P<0 small middle dot001). Mice immunized with the CPS-ovalbumin conjugate did not exhibit a reduction in mycoplasmaemia. The bactericidal activity of rabbit MmmSC-antiserum in an in-vitro growth inhibition test was related to the CPS antibody titre. This was not observed with antisera from the vaccinated mice. None of the mouse antisera exhibited growth inhibiting activity, irrespective of a high CPS or protein antibody titre (CPS-ovalbumin or WID vaccine groups, respectively). Thus, it would seem that protection against an MmmSC-induced mycoplasmaemia in the mouse is based upon cell-mediated rather than humoral immunity. The results suggest that conjugation to ovalbumin significantly increases the antibody response to CPS in the mouse; the lack of bactericidal activity of mouse anti-CPS as compared with rabbit anti-CPS in vitro suggests either that the titre of growth inhibiting antibodies is lower in the mouse or that the mechanism of growth inhibition differs between antibodies of the two species., (Copyright Harcourt Publishers Ltd.)

An indirect ELISA, based on the specific and strongly antigenic recombinant peptide of the N'-terminal half of the lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides small colony type (SC) was developed for the detection of antibodies to M. mycoides subsp. mycoides SC. It was evaluated for its suitability for serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP). The recombinant peptide containing poly-histidine residue tails was expressed in Escherichia coli and subsequently purified by Ni(2+) chelate affinity chromatography to be used as antigen to coat microtiter ELISA plates. The specificity of the antigen was tested against rabbit hyperimmune sera directed against related Mycoplasmas of the M. mycoides cluster and with sera from cattle that were either free of CBPP, but suffered from other mycoplasmal infections such as M. bovis, or showed cross-reactions in the complement fixation test. The sensitivity of the ELISA was assessed with sera from artificially infected animals and with sera from cattle originating from areas where CBPP was endemic at the time of blood sampling. The study revealed that the ELISA was both specific and sensitive for CBPP positive bovine sera and was shown also to be robust to harsh climatic conditions.

Between 1990 and 1993, 61 outbreaks of contagious bovine pleuropneumonia (CBPP) were reported in Lombardy, Northern Italy. In this study, gross pathological examination was carried out on 3129 slaughtered cattle, 716 of which (22.9%) showed typical CBPP pulmonary lesions. Single or multiple renal infarcts at different stages of development were observed in 88 (12.2%) of these 716 cattle. The kidneys of 77 cattle whose lungs showed typical CBPP lesions and were bacteriologically and immunohistochemically positive for the small colony type of Mycoplasma mycoides subspecies mycoides (M. m. mycoides SC) were selected and submitted to histological, immunohistochemical and bacteriological examination. Histologically, in chronic CBPP cases, infarcts were characterized by fibrosis, calcification of cortical tubules and tubular atrophy, accompanied by the presence of interstitial inflammatory infiltrates composed of lymphocytes, plasma cells and histiocytes. M. m. mycoides SC antigen was detected immunohistochemically in 65 (84.4%) of the 77 kidneys examined. The antigen was detected in the lumen of blood vessels and in glomerular cells. Immunolabelled interstitial cells and tubular epithelial cells were seen in chronic cases only. M. m. mycoides SC was isolated from the kidneys of 12 animals (15.6%) and more frequently in cases with renal infarcts. This study confirms previous observations that demonstrated a renal involvement in cases of CBPP. Moreover, the immunohistochemical results indicated that M. m. mycoides SC antigen was frequently detectable in different renal structures and cells in spontaneous cases of CBPP., (Copyright Harcourt Publishers Ltd.)

An abattoir study on the prevalence of contagious bovine pleuropneumonia (CBPP) in five cattle-producing states of Nigeria from 1988 to 1997 was carried out. A total of 1,936,015 slaughtered cattle was examined for characteristic CBPP lung lesions. The overall lesion-based prevalence of CBPP was 0.29% (95% CI 0.24, 0. 35). The prevalence varied significantly (P<0.05) by state but not across the years. A total of 279 CBPP outbreaks occurred and overall vaccination coverage was only 9.7%, both varied over the years and across the states. The reasons for inadequate vaccination coverage for CBPP as well as the need for re-establishment of a national CBPP control programme are suggested.

A specific PCR test for the identification of the vaccine strains T1, T1/44 and T1sr, was developed. This PCR reaction is based on variations of DNA sequences in a region flanking one IS1296 copy. The specific primer pair MmmSCP1-T1M2 amplifies a 700-bp long DNA fragment in the T1 vaccine strains and gives no amplification with the 60 other Mycoplasma mycoides subsp. mycoides SC strains tested. This PCR will permit to distinguish the T1 strain from all other vaccine strains and therefore avoid possible confusions. In addition, it should enable better investigations of post-vaccinal reactions., (Copyright 2000 Academic Press.)

Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine Mycoplasma mycoides subsp. mycoides small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for M. mycoides subsp. mycoides small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.

In 1990 an outbreak of contagious bovine pleuropneumonia (CBPP) occurred in Italy. Subsequent surveillance for CBPP was based on random sampling in bovine herds, serological controls on all animals moved from the herd of origin and controls on slaughtered animals. Official tests employed were the complement fixation test (CFT) and bacteriological isolation and typing. A total of 33,856 serum samples collected from herds in CBPP-free regions were used to define CFT specificity, while samples from 595 animals from infected herds were employed to define the sensitivity. Ninety-nine animals from three infected herds were used to estimate the sensitivity of the isolation technique. Results showed the specificity of CFT (threshold +1:10) to be 98% and sensitivity to be 63.79%. The sensitivity of the test did not change significantly, regardless of whether the lesions were caused by acute or chronic infection. The sensitivity of the isolation technique was 54.1%.

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides s.c. were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides s.c. membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.

Contagious bovine pleuropneumonia is a serious threat to cattle not only in Africa but also in southern Europe and possibly Asia. It is now present in countries that had been free of the disease for many years, giving rise to doubts about the efficiency of the control strategies. In Africa CBPP is controlled mainly by a vaccination policy that uses variant strains of Mycoplasma mycoides subsp mycoides biotype SC, called T1/44 or T1sr. Until recently, it was not possible to differentiate the various strains within the biotype and consequently to identify the vaccine strains. Restriction analysis of mycoplasma DNA with HindIII and Pst1 has been applied to 24 strains of African origin and one European strain. Each enzyme gave rise to different restriction profiles and the combination of the results permitted subdivision of these strains into 9 groups. Interestingly, some profiles of pathogenic strains seem to be restricted to certain geographical areas. The profile of the poorly immunogenic vaccinal strain KH3J is also very peculiar, and it is easily distinguished from that of the other vaccine strains originating from T1. This technique is simple once the strains are isolated. Efforts are now under way to use molecular tools based on PCR products to alleviate the difficulty of isolation.

The course of immune reactions of the manifold antigens of Mycoplasma mycoides subsp. mycoides small colony type (SC) was analysed in serum and bronchial lavage of cattle experimentally infected with the African strain Afadé and the European strain L2 using Western-blots and complement fixation. Western-blot analysis of total antigens of both strains with sera from animals infected with the homologous and heterologous strain revealed the common dominant immunogenic antigens with the molecular masses of 110, 95, 85, 80, 72, 62, 48 and 39 kDa. The sequential sampling of the blood and bronchial lavages before and after contact infections allowed us to identify the antigens of 85, 80, 72, 48 and 39 kDa as particularly early immunogens. The IgA Western blots of the bronchial lavages showed distinct, early and persistent reactions to the 110, 85, 80, 72, 48 and 45 kDa proteins. These proteins were the predominant lipoproteins as determined by [14C]palmitic acid labelling. Only relatively weak reactions of the bronchial lavages were detected with IgG. In general immune responses were significantly stronger in the animals infected with the African strain Afadé, which gave positive results two weeks after contact infection. In contrast, the animals infected with the European strain L2 induced much lower reactions with a delay of three months after contact infection. In one animal strain L2 caused no sero-conversion and no infection. The results indicate a difference in virulence between the African strain Afadé and the European strain L2.