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.     

Mycoplasma mycoides subsp. capri associated with goat respiratory disease and high flock mortality

A high mortality outbreak of respiratory mycoplasmosis occurred in goats in Mexico. The clinicopathologic presentation resembled contagious caprine pleuropneumonia caused by Mycoplasma capricolum subspecies capripneumoniae. By using a battery of polymerase chain reaction assays, the mycoplasma associated with this outbreak was identified as Mycoplasma mycoides subsp. capri.     

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.     

Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capripneumoniae.

The lppA gene, encoding the lipoprotein named LppA[Mcaca] was characterised in Mycoplasma capricolum subsp. capricolum. It encodes a lipoprotein with an apparent molecular mass of 57 kDa as determined by SDS-PAGE. Using antibodies directed against recombinant LppA[Mcaca], we showed the expression of this lipoprotein in all M. capricolum subsp. capricolum by immunoblot analysis. The serum did not cross-react with other members of the Mycoplasma mycoides cluster, hence showing that LppA[Mcaca] was antigenically specific to M. capricolum subsp. capricolum. The lppA gene was conserved within the subspecies and was used for the development of a specific PCR assay for the identification of M. capricolum subsp. capricolum. The taxonomically related Mycoplasma capricolum subsp. capripneumoniae (F38) was found to contain an lppA-pseudo-gene. It showed high similarity to functional lppA genes of other mycoplasmas in the M. mycoides cluster. However, it contained interrupted open reading frames. Moreover, the nucleotide sequence of the lppA pseudo-genes in different strains of M. capricolum subsp. capripneumoniae were quite variable. Interestingly, the lppA pseudo-gene had a size similar to that of the functional lppA genes of other mycoplasmas of the M. mycoides cluster and occupied the same genomic location as the latter ones in the vicinity of the mtlD genes. This study showed that all members of the M. mycoides cluster contain each a species-, subspecies- respectively type- specific lppA gene analogue which encodes a lipoprotein that has structural and functional relationship to the surface lipoprotein LppA [MmymySC], previously named P72, of M. mycoides subsp mycoides SC, with the exception of M. capricolum subsp. capripneumoniae which seems not to express an LppA analogue.     

山羊传染性胸膜肺炎病原的分离鉴定

从内蒙古地区山羊病料中分离出疑似山羊传染性胸膜肺炎的病原,经染色镜检、生化鉴定、血清学等方法初步鉴定为山羊支原体山羊肺炎亚种.参照已知山羊支原体ADI基因设计的引物进行PCR扩增,通过电泳分析得到与文献报道相一致的大小为316 bp的条带,最终确定为山羊支原体山羊肺炎亚种.     

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.     

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.     

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.     

Studies of the origin of false positive reactions in the serodiagnosis of contagious bovine pleuropneumonia

Four groups of cattle were experimentally immunised by four mycoplasma species of "mycoides-like" group, Mycoplasma (M) capricolum, M. mycoides subsp. mycoides (LC), M. mycoides subsp. capri and M. species group 7 of LEACH (PG50). They were then bled weekly during 2 months to establish antibodies kinetics against homologous and heterologous antigens. The standard method of complement fixation test (CFT) used in Europe and a new ELISA test for diagnosis of contagious bovine pleuropneumonia were performed in comparison with passive haemagglutination test (PHA) against antigens used for experimental immunisation. Cross reactions obtained are rather equal to the degree of similitude between these mycoplasma species. With CFT-cross reactions are transitory and occur only while homologous titers are very high, particularly with "PG50" and the two caprine mycoides strains. ELISA results using a threshold of positivity of optical density of 0.20, were similar to that obtained with CFT except ELISA specificity is not so different from CFT one. This experimental model could explain some natural situations.     

First isolation of Mycoplasma capricolum subsp. capricolum, one of the causal agents of caprine contagious agalactia, on the island of Lanzarote (Spain)

During an unusually long period of bad weather, several outbreaks of caprine contagious agalactia (CCA) were reported in a number of flocks on the island of Lanzarote (Canary Islands, Spain). Clinical and subclinical mastitis in lactating goats and some cases of arthritis and pneumonia in kids were observed in the affected flocks. Mycoplasma capricolum subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with M. mycoides subsp. mycoides "large colony type" (Mmm LC) in two flocks. This is the first report of an isolation of M. capricolum subsp. capricolum on the island of Lanzarote. The finding is of epidemiological importance and could complicate plans to control the disease. The significance of this mycoplasma species in association with CCA must now be studied in detail.     

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.     

山羊传染性胸膜肺炎研究进展

山羊传染性胸膜肺炎是由山羊支原体山羊肺炎亚种引起的一种山羊常见的高度接触性呼吸道传染病,有传染快速,发病率、死亡率高的特点,给山羊养殖业带来巨大损失。本文从病原学、流行病学、临床症状以及诊断与防控方面对山羊传染性胸膜肺炎进行综述,帮助读者更加全面、深入的了解本病,以期为山羊传染性胸膜肺炎的诊断和防治提供新思路。     

Experimental contagious caprine pleuropneumonia: a long term study on the course of infection and pathology in a flock of goats infected with Mycoplasma capricolum subsp. capripneumoniae

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in parts of Africa and Asia. It classically causes acute high morbidity and mortality early in infection, but little is known of its long term epizootiology and course. In this study, 10 goats were inoculated with Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and then mixed with 15 goats for contact transmission. The disease course was monitored in each goat for 56-105 days, whereafter the goats were killed and necropsied. Varying features signifying infection occurred in altogether 17 goats (7 inoculated, 10 in-contact). Clinical signs were severe in 8 goats but no fatalities occurred. Only 6 goats had serum antibody titres against M. capripneumoniae in ELISA. Fourteen goats (5 inoculated, 9 in-contact) had chronic pleuropulmonary lesions compatible with CCPP at necropsy and 7 of those showed M. capripneumoniae antigen in the lung by immunohistochemistry. Neither cultivation nor PCR tests were positive for the agent in any goat. The results indicate that the clinical course of CCPP in a flock may be comparatively mild, M. capripneumoniae-associated lung lesions may be present at a late stage of infection, and chronic infection may occur without a significant serological response.     

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.     

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.     

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.     

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.     

应用双重PCR方法检测羊支原体肺炎病原

通过对丝状支原体山羊亚种(M.mycoides subsp.capri,Mmc)特异性引物MmcF/MmcR和绵羊肺炎支原体(M.ovipneumontiae,Mo)特异性引物LmF/LmR退火温度、引物浓度比例等条件的选择,建立了一个可以同时检测Mmc和Mo的双重PCR方法。该方法可同时扩增出Mmc 195 bp和Mo 361 bp目的片段,但对其他病原菌不能扩增出任何条带,具有良好的特异性。敏感性试验表明,该方法能够分别检测出0.1ng的Mmc DNA和0.01 ng的Mo DNA,或同时检测出1ng Mmc和1ng Mo混合的DNA。用该双重PCR方法可对实验室保存的4株绵羊肺炎支原体和2株丝状支原体山羊亚种进行准确鉴定,并可从临床病料中检测出相应支原体,表明建立的双重PCR方法可用于Mmc和Mo的快速鉴定、实验室诊断和病原学调查。     

Contagious caprine pleuropneumonia outbreak in captive wild ungulates at Al Wabra Wildlife Preservation, State of Qatar.

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a highly contagious and serious respiratory disease of domestic goats, characterized by coughing, severe respiratory distress, and high mortality rates. The lesions at necropsy are mainly a fibrinous pleuropneumonia with increased straw-colored pleural fluid. An outbreak of CCPP in wild goat (Capra aegagrus), Nubian ibex (Capra ibex nubiana), Laristan mouflon (Ovis orientalis laristanica), and gerenuk (Litocranius walleri) occurred at Al Wabra Wildlife Preservation in the State of Qatar. The disease was suspected because of the clinical symptoms and the necropsy findings and was confirmed by the isolation and identification of the causative organism. This new finding indicates that CCPP should be considered a potential threat to wildlife and the conservation of endangered ruminant species, especially in the Middle East, where it is enzootic because of its presence in chronic carriers. Susceptible imported animals should be quarantined and vaccinated. The preferred samples for diagnosis are the pleural fluid, which contains high numbers of Mycoplasma, and sections of hepatized lung, preferably at the interface of normal and diseased tissues. Samples must be shipped to diagnostic laboratories rapidly, and appropriate cool conditions must be maintained during shipping.