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.     

Relationships between members of the Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.     

Electrophoretic analysis of proteins from Mycoplasma capricolum and related serotypes using extracts from intact cells and from minicells containing cloned mycoplasma DNA

The acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuropneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells. The organisms investigated were Mycoplasma mycoides subsp. mycoides (PG1), M. mycoides subsp. mycoides (Y-goat), M. mycoides subsp. capri (PG3), M. capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38). The results suggested a close relationship between M. capricolum and F38 and a similarly close relationship between the different M. mycoides subspecies, whereas the two M. mycoides subspecies appeared to be quite distant from M. capricolum and F38. The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the three strains of M. mycoides. Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels. In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immunoprecipitation.     

The origin of the 'Mycoplasma mycoides cluster' coincides with domestication of ruminants

The 'Mycoplasma mycoides cluster' comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the 'M. mycoides cluster'. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the 'M. mycoides cluster' dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.     

Numerical analysis of PAGE protein patterns and the taxonomic relationships within the 'Mycoplasma mycoides cluster'

Twenty-six isolates belonging to the 'Mycoplasma mycoides cluster' have been characterized by one-dimensional SDS-PAGE of their cellular proteins. A numerical classification based on the resulting patterns and using a correlation coefficient revealed four distinct phenons at a similarity (S) level of 70%, comprising: (a) bovine group 7 strains; (b) M. capricolum and F38-like strains; (c) M. mycoides subsp. capri and LC strains ('subsp. mycoides'); (d) M. mycoides subsp. mycoides (SC). At the 75% S level, they could be divided further to give eight phenons. The composition of the clusters at both levels was in good agreement with their previous classification, except for M. mycoides subsp. mycoides LC and M. mycoides subsp. capri, which were clustered in a single phenon at 70% S and could not be clearly separated at 75% S. We conclude that high-resolution SDS-PAGE, combined with computerized analysis of protein patterns, provides an extremely effective approach to the investigation of taxonomic relationships within this group of mycoplasmas.     

Development of two PCR assays for the identification of mycoplasmas causing contagious agalactia

Abstract A new detection test for the mycoplasmas causing contagious agalactia, Mycoplasma agalactiae , M. capricolum subsp. capricolum and M. mycoides subsp. mycoldes L.C., was developed. It was based on two polymerase chain reaction assays: the Ma-PCR for the detection of M. agalactiae and the MYC-PCR for the 'mycoides cluster' thus including M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. An M. agalactiae strain was identified by a 933-bp Ma-PCR product and no amplification with the MYC-PCR. In contrast, a 460-bp MYC-PCR product and a negative or a 350-bp Ma-PCR product characterized a 'mycoides cluster' strain. M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. were identified by their species-specific Asel pattern of the 460-bp MYC-PCR product.     

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.     

Electrophoretic Analysis of Isoenzymes of Mycoplasma Species

The purpose of this paper is to further characterize and determine relatedness among some Mycoplasma species or serogroups of significant practical veterinary interest. Twenty-four strains were examined for the presence of 35 enzymes by horizontal starch gel electrophoresis, revealing a total of 127 different electromorphs of 30 enzymes. Inter- as well as intraspecific differences were found demonstrating the application of isoenzyme studies in classification as well as epidemiology. It is concluded that the F38 group of MacOwan and group 7 of Leach constitute 2 new species, The elevation of the 2 subspecies of M. mycoides (mycoides and capri) to species level is favoured, but it is suggested that a decision be taken internationally, considering the practical consequences of a given nomenclature. Three alternative possibilities for classification are presented. Regarding identification the results suggest that the presence or absence of maltase and ornithine transcarbamylase indicate whether an isolate is related to the agent of contagious bovine pleuropneumonia or merely a representative of either the caprine LG type of M. mycoides subsp. mycoides or the classical caprine subspecies, M. mycoides subsp. capri.     

Isolation of Mycoplasma mycoides, mycoides (LC variant), from two naturally aborted caprine fetuses

Described in this study are 2 cases of caprine abortion associated with the LC type of Mycoplasma mycoides , subsp. mycoides . This mycoplasma and Mycoplasma mycoides , subsp. capri had been previously reported in adult goats in this herd. The abortion took place in the latter part of gestation. Samples from heart blood, lung and pleural exudate were collected for the isolation of mycoplasmas and other bacterials in both fetuses. Two strains of Mycoplasma mycoides , subsp. mycoides (LC variant) were isolated. The only gross lesion of the internal organs in the aborted fetuses was congestion of the lungs. Microscopic lesions were encountered in the lungs, and these were characterized by patchy to diffuse pneumonia. Exfoliated cells, many alveolar macrophages, scattered neutrophils and lymphocytes were seen in the lumen of the terminal airways and alveolar spaces. This report appears to be the first isolation of Mycoplasma mycoides , subsp. mycoides (LC variant) from aborted caprine fetuses.     

Taxonomic significance of differences in DNA methylation within the 'Mycoplasma mycoides cluster' detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ' Mycoplasma mycoides cluster' was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI , which is inhibited by the methylation, or DpnI , which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum , plus the F38-like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.     

One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains

The ability to utilize maltose, as determined by measurement of oxygen uptake, is used to differentiate Mycoplasma mycoides subsp. mycoides small colony (SC) and M. capricolum subsp. capripneumoniae (all strains negative) from other members of the M. mycoides cluster (M. mycoides subsp. capri, M. mycoides subsp. mycoides large colony (LC), M. capricolum subsp. capricolum; and bovine serogroup 7; 94% of strains positive). Rapid tests for maltose utilizing ability were developed, based on hydrolysis of a chromogenic alpha-glucosidase (maltase) substrate (p-nitrophenyl-alpha-D-glucopyranoside, colourless) to give a brightly coloured product (p-nitrophenol, yellow). On agar plates, colonies of maltose-utilizing strains became coloured within 40 min.     

Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding beta-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli beta-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.     

山羊传染性胸膜肺炎病原体4株国内分离株的重新分类

山羊传染性胸膜肺炎(Contagious Caprine Pleuropneumonia,CCPP)是由山羊支原体山羊肺炎亚种(M.capricolum subsp.capripneumoniae,Mccp)引起的高度接触性传染病,对4株CCPP中国分离株进行分子特征研究,确定其分类地位。针对3段基因(A、B、C),对扩增产物进行酶切鉴定和测序,将结果与丝状支原体簇的6个成员进行遗传衍化分析。在A片段,4株中国分离株的扩增产物经PstI酶切后的结果与Mccp代表株F38相同,为548、420、128等3条带,其他5个丝状支原体簇成员只有420、128bp两条带。在B片段,序列分析结果显示4株中国分离株与F38同源性为99.5%,与山羊支原体山羊亚种Mcc代表株kid的同源性为98.9%,与丝状支原体山羊亚种MmcZZ株同源性仅为95.4%。在C片段研究发现,4株中国分离株的序列与Mmc模式株PG3株同源性为67.4%~67.6%,与2株Mcc8601-50和California Kid同源性为95.1%~98.4%,与3株Mccp97097ET、Gabes和F38的同源性为99.6%~99.8%。通过对中国分离的87001、87002、367、1653等4株CCPP病原体的分子特征研究,首次提出其与山羊支原体山羊肺炎亚种(Mccp)亲缘关系最近,应归属为Mccp,并将国内流行的山羊传染性胸膜肺炎的病原定名为Mccp。     

The F38-Like Group,a New Group of Caprine Mycoplasmas?

This paper concerns the taxonomic status of the F38-like group (MacOwan), a prime determinant of contagious caprine pleuropneumonia (CCPP). Extensive biochemical and serological investigations on strain F38 are reported. Some complex serological relationships with other mycoplasma species are revealed. The results, taken in conjunction with earlier published work on geno-typic characters, lead to the conclusion that final classification of these organisms should await further comparative studies of a number of field strains with a related group of strains classified as M. capri-colum. The characterization of F38 confirms its partial relationship to the “M. mycoides group” of ovine/caprine/bovine mycoplasmas, and has also revealed a very close phenotypic relationship to the bovine mycoplasma serogroup 7, a finding of potential diagnostic and epidemiological importance.     

Comparison of ureaplasmas from sheep and goats with Ureaplasma diversum and U. urealyticum

Ureaplasmas isolated from sheep and goats were compared by immunofluorescence with antisera prepared in calves and by PAGE of polypeptides labelled by growth in the presence of [35S]methionine. The ovine and caprine strains constituted two groups defined by serology and polypeptide composition that were not related to the animal species from which they originated. Strains representing these two groups were compared with Ureaplasma urealyticum (human isolates) and U. diversum (bovine isolates). They could not be classified with either but were more similar to the U. diversum strains.     

Mycoplasmas: Use of Polyvalent Antisera for Identification by Indirect Immunofluorescence

It would be an advantage, under many circumstances, to be able to make use of polyvalent antisera in the process of identifying mycoplasmas. As the indirect immunofluorescence test is sufficiently sensitive and also generally accepted as being rather specific, this technique was chosen to investigate whether polyvalent antisera are applicable in routine identification of mycoplasmas. Three polyvalent sera were used, each consisting of 9 or 10 rabbit antisera raised against 29 of the more common species of the genus Mycoplasma. Twenty-six field strains were examined. One strain did not react with any of the 3 polyvalent antisera although it was later identified as M. bovigenitalium. The remaining 25 strains reacted with 1 and only 1 of the polyvalent antisera and were subsequently identified by immunofluorescence utilizing monospecific antisera. Strains of the following species were identified: M. arginini, M. bovigenitalium, M. bovis, M. bovoculi, M. canis, M. capricolum, M. cynos, M. edwardii, M. hominis, M. hyorhinis, M. molare, M. mycoides subsp. mycoides and M. opalescens. It is concluded that polyvalent antisera may be used in identification procedures and thereby permit the use of a limited number of monospecific antisera without preceding biochemical testing.     

Genome sequence of Mycoplasma capricolum subsp. capripneumoniae strain M1601

Mycoplasma capricolum subsp. capripneumoniae is the causative agent of contagious caprine pleuropneumonia, a devastating disease of goats listed by the World Organization for Animal Health. Here we report the first complete genome sequence of this organism (strain M1601, a clinically isolated strain from China).