Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction

Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.     

Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.

Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.     

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.     

Extended surveillance for CBPP in a free country: Challenges and solutions regarding the potential caprine reservoir

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease of cattle and buffalo caused by Mycoplasma mycoides subsp. mycoides "Small Colony" (MmmSC). The agent of CBPP has been isolated from goats in different countries including CBPP-free areas. Goats can therefore be regarded as a putative MmmSC reservoir. No diagnostic test for CBPP surveillance in goats has been proposed as yet. Furthermore, serological tests could be seriously hampered by a widespread caprine infection due to the subspecies M. mycoides subsp. capri (Mmc), which is antigenically very close to MmmSC and displays high levels of genetic variability. A competition ELISA (cELISA) is currently used to screen for CBPP in cattle at the herd level in infected areas. The aim of this study was to see if the same cELISA would be specific enough to be used to screen goats despite the potential concomitant infection with Mmc. The cELISA titers of goats from Mmc-infected and non-infected herds were comparable and negative using the accepted cutoff for bovine sera. In contrast, seroconversion was observed in goats experimentally inoculated with an Mmc strain that cross-reacted with a monoclonal antibody targeting the same epitope as that used in cELISA. The probability of such false positivity occurring under field conditions is very low since Mmc strains with such an atypical antigenic profile emerge only rarely as a result of random nucleotide variation of the epitope-coding region. In conclusion, the commercially available cELISA can be considered specific enough to be used as a primary test to monitor passage of the CBPP agent in goats, but its sensitivity in goats requires further investigation.     

Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC.

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.     

Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species

We describe the development and analytical validation of a 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for detection of multiple ruminant Mycoplasma spp. The assay employs a combination of newly designed and previously validated primer-probe sets that target genetic loci specific for Mycoplasma bovis, Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subspecies capripneumoniae (Mccp). Analytical sensitivity for the targeted Mycoplasma species ranged from 10 fg to 1 pg of purified gDNA extracted from broth cultures (approximately 8-800 MmmSC genome equivalents). In silico comparison of primers and probes, and analytical assessment with a range of near-neighbor Mycoplasma species and multiple bacterial respiratory pathogens demonstrated 100% analytical specificity of the assay. To assess assay performance and diagnostic specificity, 192 bovine respiratory samples were analyzed by incorporating a high throughput DNA extraction platform. The assay correctly classified all samples as negative for MmmSC or Mccp. All 33 field samples confirmed as positive for M. bovis by sequencing the uvrC gene were positive in the assay. The results from this study indicate that the bead-based liquid suspension array will provide a reliable, analytically sensitive and specific platform to simultaneously interrogate ruminant respiratory samples for multiple Mycoplasma species, including M. mycoides cluster organisms that are exotic to the United States. Sequential addition of primer-probe sets to the assay did not significantly impact analytical sensitivity of individual primer-probe combinations, suggesting that expanding the assay to include more Mycoplasma species will not compromise overall performance.     

Detection of Mycoplasma mycoides subspecies mycoides SC in bovine lung and lymph node tissues by culture, sandwich ELISA and polymerase chain reaction systems

Cattle from Northern Portugal, many with pulmonary lesions typical of contagious bovine pleuropneumonia, were investigated for the presence of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), which is the causative agent of CBPP, with several detection tests. Sandwich ELISA that included a culture enrichment stage, and 2 different PCR diagnostic systems were used to detect MmmSC in lung and mediastinal lymph node tissues from these animals. The comparison of typical CBPP pathology with the results of detection revealed that no single one of these methods provided a perfect match to the pathological data. Best performing tests were the PCR with laser induced fluorescence and PCR with pleuroTRAP kit (Chemicon, Australia), which are diagnostic systems based on amplification of genomic MmmSC DNA followed by sensitive detection of the amplified products. These were followed by the broth-enriched sandwich ELISA, which uses a monoclonal antibody specific to the M. mycoides cluster, to capture the antigen.     

Clinical, humoral and IFNgamma responses of cattle to infection with Mycoplasma mycoides var. mycoides small colony and attempts to condition the pathogenesis of the infection

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 na?ve 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--a nearly forgotten disease?

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) is a notifiable disease and has to be reported to the World Organisation for Animal Health (Office International des Epizooties, OIE, http://www.oie.int). Despite the fact that the last reported cases in Germany date back to 1926, the risk of introduction through clinically inconspicuous animals from countries where the disease is still endemic is rising. This is due mainly to an increase in international trade of live cattle and the failure to contain CBPP in many parts of Africa and elsewhere. To detect and eliminate this highly contagious infectious disease of the bovine respiratory tract, it is necessary to recognize matching clinical symptoms as soon as possible, as well as to have efficient methods for its detection on hand. In the present paper, we describe clinical manifestations and review state-of-the-art research, as well as currently used detection methods.     

Analysis of cellular responses to Mycoplasma mycoides subsp. mycoides small colony biotype associated with control of contagious bovine pleuropneumonia

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.     

Danofloxacin (Advocin) reduces the spread of contagious bovine pleuropneumonia to healthy in-contact cattle

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most important diseases of cattle in Sub-Saharan Africa. The live T1/44 vaccine is normally used for its control but produces only transient protection and gives rise to adverse reactions. The present study evaluated the efficacy of danofloxacin (2.5% Advocintrade mark, Pfizer Ltd.) for the treatment of naturally infected cattle and in the prevention of CBPP transmission to in-contact cattle. Adult cattle, taken from a natural outbreak, were placed into two groups of 10 animals and kept on a research farm in paddocks 50m apart. One group was treated with 2.5mg/kg danofloxacin on days 0, 1 and 2; the other group were saline treated. On day 2, 10 CBPP-free, seronegative cattle were placed in contact with each of the two groups. All cattle were monitored for 3.5 months. No differences were seen in clinical improvement of the CBPP-affected cattle treated with danofloxacin compared with the untreated CBPP-affected cattle with approximately half of each group being withdrawn because of CBPP or showing CBPP lesions at post mortem examination. Clinical scores of the two groups were also similar. However cattle kept in contact with the danofloxacin-treated CBPP-affected animals showed significantly fewer lesions, less mortality and fewer animals were seropositive (P<0.02) and had reduced clinical scores (P<0.001) compared to cattle kept in contact with untreated CBPP-affected cattle. MmmSC was also isolated from fewer contact controls kept with the treated group. These findings could have important implications for the control of CBPP in Africa.     

Characterisation of the lymph node immune response following Mycoplasma mycoides subsp. Mycoides SC infection in cattle

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.     

Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.     

Within-herd spread of contagious bovine pleuropneumonia in Ethiopian highlands

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).     

Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC

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.     

Indirect infection of cattle with contagious bovine pleuropneumonia.

Hay infected with Mycoplasma mycoides sub-species mycoides (M mycoides) was fed to six animals on three occasions. Five animals developed complement-fixing antibody in their sera and gave a positive reaction to the comparative intradermal allergic test. No animal died from contagious bovine pleuropneumonia (CBPP) but when they were killed, three had unequivocal lesions of the disease. M mycoides was isolated from the animals with these lesions but not from the others. CBPP was transmitted to two animals put in contact with those animals showing disease, and M mycoides was recovered from lesions in both animals. It is suggested that when unexplained outbreaks of CBPP occur, the possibility of indirect transmission should be considered.     

Gamma interferon-producing CD4 T-cells correlate with resistance to Mycoplasma mycoides subsp. mycoides S.C. infection in cattle

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.     

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.     

Mycoplasmas isolated from the respiratory tract of cattle and goats in Tanzania

A microbiological study of the mycoplasma flora in the respiratory tracts of cattle and goats in selected regions of Tanzania is described. In the examination of cattle, mycoplasmas were isolated from 60 (17.8%) of the 338 examined lung samples, 8 (47.1%) of the 17 lymph nodes, 4 (13.3%) of the 30 pleural fluid samples and 4 (3.9%) of the 103 nasal swabs examined. All the isolates were identified as Mycoplasma mycoides subsp. mycoides, Small Colony type except for one isolate from pleural fluid which was identified as Mycoplasma arginini. M. mycoides subsp. mycoides, Small Colony type was isolated from samples originating from Dodoma, Iringa, Mbeya, Morogoro and Shinyanga regions where outbreaks of contagious bovine pleuropneumonia had been reported. In the examination of goats, mycoplasmas were isolated from 54 (34.0%) of the 159 examined lung samples, 41 (18.1%) of the 226 nasal swabs and 4 (40.0%) of the 10 pleural fluid samples. The species demonstrated were Mycoplasma capricolum subsp. capripneumoniae, M. mycoides subsp. mycoides, Small Colony type Mycoplasma ovipneumoniae and M. Capricolum subsp. arginini. The isolation of M. capripneumoniae in the Coast and Morogoro regions confirmed the presence of contagious caprine pleuropneumonia in the regions.