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。     

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.     

Molecular epidemiology of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides subspecies mycoides biotype SC strains

Contagious bovine pleuropneumonia is a bacterial disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), and included in list A of the Office International des Epizooties. It is one of the major constraints to cattle raising in sub-Saharan and south-western Africa and also a threat to all countries currently free of the disease. MmmSC strains were considered very homogeneous until 1995, when various techniques such as enzymatic restriction of whole DNA or Southern blotting showed that this was not the case. These techniques are unfortunately difficult to standardize and require the extraction of DNA from an MmmSC culture. We therefore decided to investigate the possibility of constructing a molecular epidemiology tool based on multilocus sequence analysis (MLSA) with PCR amplification of various loci followed by sequencing. Six loci were found suitable for this purpose and an additional PCR was designed to detect the presence of an 8.8kb deletion described by others in some strains. Fifteen different MLSA profiles were evidenced in our study. They allowed a clear distinction between European, south-western African and sub-Saharan strains. In addition, the results obtained on strain PO1967 confirmed its European origin, even though it does not exhibit the 8.8kb deletion. This new tool for contagious bovine pleuropneumonia may prove particularly useful for identifying MmmSC strains in countries at risk from contamination. It can also easily be refined by adding more strains or other loci of interest.     

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

Lipoprotein lppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP). The lppC gene was found in the type strain of M. mycoides subsp. mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M. mycoides subsp. mycoides SC, of which only one seems to be functional. Genes homologous to lppC have also been detected in closely related mycoplasmas such as M. mycoides subsp. mycoides large colony (LC) type and in M. sp. bovine group 7. lppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II. The amino acid sequence of lppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M. mycoides subsp. mycoides SC. The N-terminal domain of the mature lppC seems to be surface exposed. The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane. A recombinant peptide representing the N-terminal half of lppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts. The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M. mycoides subsp. mycoides SC.     

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.     

丝状支原体丝状亚种SC生物型PCR检测方法的建议

建立了一个可以识别丝状支原体丝状亚种SC生物型（MmmSC)的PCR方法。根据丝状支原体丝状亚种SC生物型（MmmSC)相关核苷酸序列，设计合成了MC1、MC2和SC1、SC2两对引物。MC1、MC2是一对簇特异性引物，用于鉴别丝状支原体族6个成员，对MmmSC、MmmLC扩增出与预期结果相符的462bp片段；而SC1、SC2是针对MnnSC的一对特异性引物，只能对MmmSC扩增出277bp的片段，经过VspI、Bsp143I和Dral三种限制性内切酶鉴定与预期结果相符，而不能扩增出MmmLC型Y－goat代表株，说明具有非常好的特异性。对MmmSC进行的敏感性试验显示SC1、SC2引物能够检测到100个CFU，具有非常高的敏感性。     

Application of dot immunobinding on membrane filtration (MF dot) to the study of relationships within "M. mycoides cluster" and within "glucose and arginine-negative cluster" of ruminant mycoplasmas.

A total of 189 isolates from cattle, sheep and goats, allocated to two groups on biochemical grounds, have been examined by a dot immunobinding membrane-filtration (MF dot) method. Seventy glucose fermenting isolates, showing relationships with the "Mycoplasma mycoides cluster", have been compared by MF dot against polyclonal hyperimmunesera prepared against the following reference strains: M. mycoides subsp. mycoides, small colony type (SC), strain PG1 and large colony type (LC) strain Y Goat; M. capricolum strain California Kid (CK); M. species bovine serogroup 7 strain PG50, and, ovine/caprine serogroup 11 strain 2-D. The isolates fell into 5 main groups: (a) 14 serologically homogeneous isolates similar to subsp. mycoides SC PG1 (b) 4 homogeneous isolates similar to PG50 (c) 14 isolates all serologically similar to Y Goat, but heterogeneously reactive with subsp. capri PG3 and M. capricolum CK antisera (d) 7 isolates serologically similar to subsp. capri PG3, but heterogeneously reactive with subsp. mycoides SC PG1, M. capricolum CK and 2-D antisera (e) 28 isolates strongly reactive with both M. capricolum CK and serogroup 7 PG50 antisera. 119 isolates that were all glucose and arginine negative were also compared by the MF dot method with the reference strains. Most of these could be classified definitely as M. bovis (78 isolates), M. agalactiae (21 isolates) and serogroup 11 (5 isolates). 13 isolates gave a strong reaction with both M. bovigenitalium and serogroup p11 antisera. 2 isolates showed an unclassifiable pattern. The results confirm that the glucose and arginine-negative cluster strains that reacted with 2-D antiserum, also share serological relationships with the "M. mycoides cluster", albeit with a very heterogeneous pattern.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC 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 SC 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.     

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.     

SUBDIVISION OF MYCOPLASMA MYCOIDES SUBSP. MYCOIDES FROM CATTLE AND GOATS INTO TWO TYPES

Some strains of Mycoplasma mycoides subsp. mycoides, mostly isolated from goats, grow to greater turbidity in broth and form larger colonies on solid medium than does the type strain, PG1. These strains also digest casein, liquefy inspissated serum actively and survive longer at 45 degrees C and are referred to as LC (large colony) strains. Strains more closely resembling PG1 have been called SC (small colony) strains. The SC strains comprise those from contagious bovine pleuropneumonia (CBPP) and some from goats. One LC strain was isolated from a steer; all others have come from goats. Differentiation of M. mycoides subsp. mycoides into 2 types on the basis of the characteristics described may be relevant to their role in CBPP.     

丝状霉形体丝状亚种SC型脂蛋白LppQ N末端基因克隆与序列分析

脂蛋白LPPQ是丝状霉形体丝状亚种SC型(MmmSC)非洲株、欧洲株和疫苗株所特有的。LPPQ N末端域具有良好的免疫原性，在牛体内可诱导产生强大、特异、早期、持续的免疫反应。本研究根据已发表的LPPQ基因序列设计引物，用Pyrobest^TM高保真DNA聚合酶从MmmSC HVRI X株中扩增出了LPPQ N末端基因序列，并进行了克隆与序列测定。核苷酸序列比较结果显示，HVRI X株的LPPQ N末端基因序列与国外发表的序列同源性为99．7％，由其推导的氨基酸序列同源性为99，1％，为脂蛋白LPPQ N末端基因体外表达奠定了基础。     

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.     

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.     

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.     

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.     

Rapid screening of H(2)O(2) production by Mycoplasma mycoides and differentiation of European subsp. mycoides SC (small colony) isolates

Mycoplasma mycoides strains were screened for the ability to produce H(2)O(2) from glucose and glycerol metabolism using rapid and simple colorimetric assays. In quantitative assays, H(2)O(2) production by washed cell suspensions was detected by the oxidation of o-dianisidine in the presence of peroxidase. In qualitative assays, a 3,3'-diaminobenzidine-peroxidase reagent was applied to colonies on agar plates. Both methods enabled differentiation of European subsp. mycoides SC (small colony) isolates from other M. mycoides strains by their inability to produce H(2)O(2) from glycerol metabolism. In addition, two strains of subsp. capri were identified which produced large amounts of H(2)O(2) from glucose oxidation. In lysed cells of these strains, NADH oxidation gave approximately 1 mol H(2)O(2) per mol NADH oxidised whereas in 36 subsp. mycoides and 10 other subsp. capri strains, the quantity produced was 0.01-0.20mol H(2)O(2) per mol NADH oxidised.     

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.     

丝状支原体丝状亚种SC型脂蛋白Q的N末端定点突变及表达载体的构建

根据已发表的丝状支原体丝状亚种SC型（MmmSC）LppQ基因序列设计引物，从MmmSC HVRIx株中扩增出了LppQN末端基因，并将其分别克隆到pUC18和pUC19上，利用一步重叠延伸PCR突变方法将其66位的TGA突变为TGG，经克隆与序列测定证实突变成功后，将已突变的LppQN末端基因插入原核表达载体pET32a的多克隆位点，成功地构建了LppQN末端基因原核表达载体pET32a-LppQ，为其下一步体外表达奠定了基础。     

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.