Strains of Mycoplasma mycoides subsp. mycoides small colony type isolated in China between 1953 and 1960 show close similarity to strains of the Africa/Australia cluster

In this study, six Chinese strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated between 1953-1960 were analysed and their molecular characteristics compared to those of the African PG1 and Afade strains, the European C305 and 138/5 strains and the closely related caprine M. mycoides subsp.mycoides large colony type Y-goat strain. PCR amplification of long DNA fragments showed that the six Chinese strains, the PG1 strain and the Y-goat strain, just like Afade, did not have the 8.84 kb deletion characteristic of the European strains C305 and 138/5. In comparison, the lppB gene sequence of the six MmmSC Chinese strains was found to be 99% homologous to that of PG1and Afade, but <93% homologous to the Y-goat sequence. The anti-rLppB antiserum reacted with PG1, Y-goat and the six Chinese strains at 67 kDa sites in Western blot, indicating that the lppB gene and its encoding protein exist in the Chinese strains. Multilocus sequence analysis (MLSA) of MmmSC strains from various regions confirmed that the Chinese strains were identical to the African and Australian cluster. This finding was further supported by the outcome of selective primer amplification. Based on these results, it is suggested that CBPP in China may have originated from Australia.     

Genotyping of Mycoplasma mycoides subsp. mycoides SC by multilocus sequence analysis allows molecular epidemiology of contagious bovine pleuropneumonia

Mycoplasma mycoides subsp. mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). Although eradicated in most developed countries, the disease reappeared in Europe in the 1990s. This reappearance may have been caused either by importation from sub-Saharan Africa, where CBPP is still endemic, or by the reemergence of virulent strains in Europe, as suggested by earlier studies. A multilocus sequence analysis scheme has been developed to address this issue and, most importantly, to be able to monitor new epidemics. The alignment of the full genome sequence of the reference strain PG1 and the partial genome sequence of a pathogenic strain allowed the identification of polymorphic sites. Nineteen initial loci were selected within housekeeping genes, genes of unknown function and non coding sequences. The suitability of these loci for genotyping MmmSC strains was first tested on six strains of diverse geographic origin. The analyses showed that the published PG1 sequence contained a number of specific polymorphisms that were therefore of no use for molecular typing. Among the eight informative polymorphic loci finally selected, only one (ftsY) was positioned within a housekeeping gene. Three main groups and 31 different allelic profiles were identified among 51 strains and strain variants examined. Cluster analysis confirmed that European strains from the 1990s did not originate from Africa. It also showed a genetic link between a European strain isolated in 1967 and those found in southern Africa and Australia. This was in agreement with historical data showing that CBPP was introduced in these regions during colonisation in the 19th century.     

Identification and differentiation of European and African/Australian strains of Mycoplasma mycoides subspecies mycoides small-colony type using polymerase chain reaction analysis.

Mycoplasma mycoides subspecies mycoides small-colony type (M. m. m. SC) is the cause of the economically important contagious bovine pleuropneumonia. Isolates from Africa and Australia have previously been documented to have a fragment of approximately 8.84 kb, which is absent in European strains. A set of polymerase chain reaction (PCR) primers over this region was designed to identify M. m. m. SC isolates and separate European strains from those of Africa/Australia. Specificity of the PCR assay was achieved through the positioning of an oligonucleotide within the insertion sequence IS1296, upstream of this deletion, which then was paired with a reverse primer, upstream of the deletion, within the 8.84 kb-deleted region or downstream of the deletion, generating fragments of 1.1 kb (all M. m. m. SC strains), 1.4 kb (African/Australian strains only) and 1.3 kb (European strains only), respectively. Identification and differentiation was specific for DNA from M. m. m. SC with no amplification of DNA from other cluster members or closely related species. The PCR products did not require differentiation by use of a restriction endonuclease, and have potential for use in detection of this organism in clinical samples.     

A simplified PCR method for genotyping Mycoplasma mycoides subspecies mycoides small colony: the aetiologic agent of contagious bovine pleuropneumonia

Mycoplasma mycoides subspecies mycoides small colony is the aetiological agent of contagious bovine pleuropneumonia, a cattle disease endemic to areas of sub-Saharan Africa. Twenty isolates from various geographical locations and the type strain were analysed by multi-locus sequence analysis (MLSA). The data generated was then used to develop three PCR primer sets to differentiate these isolates. The PCRs differentiated the isolates into four groups; the type strain (T); isolates of European origin (Eu); isolates from Tanzania (Af1) with a final group consisting of isolates from Namibia and Botswana (Af2). These PCRs offers a rapid and efficient post-identification typing method without the need to sequence and analyse multiple genes.     

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.     

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.     

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.     

丝状支原体丝状亚种SC型中国分离株分子流行特征

收集了6个来自不同地方中国历史流行的丝状支原体丝状亚种SC型（Mycoplasma mycoides subsp．MycoidesSC，MmmSC）毒株与PG1、Y—goat进行分子特征比较。全茵体蛋白电泳与PG1完全相同，而与Y-goat不同；PCR结果显示6个中国分离株与PG1、Y-goat都没有缺失8．84kb片段；LppB全基因序列比较发现与PG1同源性达99％，而与Y—goat同源性很低；多位点基因序列类型（Multilocus sequence types，MST）分析显示与非洲澳大利亚群完全相同。以上证据显示中国CBPP流行株应归属于非洲澳大利亚群。     

Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony

Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.     

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type

Minimum inhibitory concentrations (MIC) and minimum mycoplasmacidal concentrations (MMC) of the antimicrobials danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin were determined in vitro for 20 isolates of Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), the causative agent of contagious bovine pleuropneumonia (CBPP). The majority of strains were most susceptible to tilmicosin, followed by danofloxacin, oxytetracycline, florfenicol and spectinomycin with MIC50 values of 0.015, 0.25, 0.5, 1 and 8 microg/ml, and MMC50 values of 0.06, 0.5, 8, 8 and 16 microg/ml, respectively. However, tilmicosin had poor mycoplasmacidal activity against two recent strains from Portugal. There was no evidence of resistance to danofloxacin in any of the strains.     

Serological survey to determine the occurrence of respiratory Mycoplasma infections in the Polish cattle population

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.     

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.     

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.     

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.     

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.     

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.     

Phylogeny of the Mycoplasma mycoides cluster as shown by sequencing of a putative membrane protein gene

The Mycoplasma mycoides cluster is made of six species that are closely related both genetically and phenotypically. Two are of particular importance, M. mycoides subsp. mycoides SC causing contagious bovine pleuropneumonia and M. capricolum subsp. capripneumoniae causing contagious caprine pleuropneumonia. The sequences of a putative membrane protein gene and partial flanking open reading frames have been obtained from various strains in this cluster, including all reference strains. Sequence analysis showed this locus is present and fully conserved in all strains of M. mycoides subsp. mycoides SC isolated from geographically most distant places worldwide. In M. capricolum subsp. capripneumoniae polymorphism in this locus has been found at seven positions and revealed that they can be used as epidemiological markers. Conserved regions were used to define a primer pair that enables the amplification by PCR of two fragments 302 and 1298bp long, respectively. The 302bp long fragment contains an intergenic sequence that can be used for phylogenetic studies or for identification purposes. Parsimony analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides cluster into two subgroups that is in accordance with results obtained by phenotypic methods. Two lineages exist within the capricolum subgroup, one of them clustering strains identified as M. capricolum subsp. capricolum, M. capricolum subsp. capricolum and M. sp Bovine Group 7. However M. capricolum subsp. capripneumoniae strains can readily be identified by three specific nucleotide positions or by sequencing the 1298bp long fragment. There is no clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides subsp. mycoides LC and M. mycoides subsp. capri should not be separated into two subspecies. Mycoplasma mycoides subsp. mycoides SC strains can easily be distinguished as they bear an insertion sequence 15bp downstream from the stop codon of the membrane protein gene.     

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.     

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.     

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.