Mycoplasma mycoides subspecies mycoides as the cause of a subauricular abscess and mastitis in a goat.

A 6-year-old female goat was admitted with right-sided subauricular swelling and facial nerve paralysis. Mastitis developed subsequently. The subauricular swelling localized to a mass and was excised. Mycoplasma mycoides subsp mycoides was cultured from samples obtained from the mass and mammary gland. After clinical improvement, the goat was discharged to the owner with instructions to isolate the goat and to submit milk samples from the rest of the herd for microbial culturing. Mycoplasma spp can spread to other tissues from an initial site of infection. In goats with clinical signs similar to those of the goat of this report, samples should be obtained for microbial culture on Mycoplasma medium. Goats infected with Mycoplasma spp should be isolated or culled because of the risk of transmission to uninfected animals.     

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.     

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

The identification of an organism isolated from maned sheep as Mycoplasma mycoides subsp. mycoides

The natural host-range of M. mycoides subsp. mycoides is generally believed to be restricted to cattle, although the production in experimental conditions of a generalized infection of sheep and goats has been reported on some occasions.     

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.     

猪肺炎支原体表面蛋白P46基因的克隆与序列比较

猪肺炎支原体(Mycoplasma Hyopneumoniae,Mhp)兔化弱毒株R659株、济南强毒株、强毒Z株、国际标准株232,通过A26培养基培养,提取DNA;利用PCR技术从四株猪肺炎支原体中均能扩增出目的条带.将该序列克隆到pGEM-T-Easy载体上测序.结果表明,克隆序列全长1 152 bp,编码383个氨基酸和一个终止子(TAA);该序列中含有3个TGA编码Trp,而不是终止密码子.比较兔化弱毒株与济南强毒株、国际标准株232及NCBI上发布的J株的P46基因序列,发现它们的同源性分别为98.6%、99.2%、99.2%;比较它们编码的氨基酸发现它们的同源性分别为98.7%、99.2%、99.2%.结果表明猪肺炎支原体的P46基因在猪肺炎支原体种内是很保守的,因此建立以P46蛋白为诊断抗原的ELISA具有潜在的意义.     

Induction of mycoplasmosis in goat kids by oral inoculation with Mycoplasma mycoides subspecies mycoides

A one-time, orally administered dose of greater than or equal to 1 X 10(6) colony-forming units of Mycoplasma mycoides subspecies mycoides was sufficient to induce clinical mycoplasmosis (n = 37) terminating in fatal mycoplasmemia in 73% (37 of 51) of the clinically affected kids. The pathogen was isolated from the blood samples as early as 24 hours after oral inoculation; hot, swollen joints frequently were evident by 4 or 5 days after exposure. Pyrexia (to 42.3 C) was detected in about 95% (35 of 37) clinically affected kids, although about 5% (2 of 35) died peracutely without fever or other premonitory signs. At necropsy, the cardinal lesions were a fibrinopurulent polyarthritis and red, patchy to diffuse areas of consolidation in 1 or more lung lobes. At death, usually within 4 to 16 days after oral inoculation, the concentration of M mycoides subspecies mycoides in the blood was 1 X 10(6) to 1 X 10(7) colony-forming units/ml. Histologically, the kids had diffuse fluid leakage into pulmonary alveoli and to a lesser extent into small vessels of various other organs. Fibrinocellular thrombi of terminal occurrence were occasionally present in various organs. The meningeal, pleural, and peritoneal surfaces had vascular leakage and a minimal perivascular accumulation of leukocytes. The disease was contagious. Of 14 noninoculated control kids in close confinement with affected kids, 8 (57%) developed mycoplasmosis in 7 to 15 days and died of mycoplasmemia. The remaining 5 noninoculated kids remained healthy, as did noninoculated kids that were kept isolated from affected kids.     

Differential clustering of Mycoplasma mycoides subsp. mycoides SC strains by PCR-REA of the bgl locus

In order to develop a specific tool differentiating the African field strains of Mycoplasma mycoides subsp. mycoides SC from other potentially less virulent strains, including the vaccine strains, we have developed a PCR followed by a restriction enzyme analysis (PCR–REA). This approach also differentiates the African field strains from the Australian strains and the type strain PG1. The genomic marker detected by the PCR–REA is based on a single nucleotide change in the bgl gene that codes for 6-phospho-β-glucosidase (Bgl), an enzyme that is involved in sugar metabolism.     

羊丝状支原体簇环介导等温扩增检测方法的建立

本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65 ℃等温条件下,60 min一步完成反应。在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色。丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应。以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL。结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法。     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.     

Inhibition of the growth of some strains of Mycoplasma mycoides subsp mycoides by the blood of certain horses.

When incorporated in solid medium at a concentration of 15 per cent, the defibrinated blood of certain horses strongly suppressed the growth of some, but not all, strains of Mycoplasma mycoides subsp mycoides so that many colonies failed to develop to a visible size. Blood from a single rabbit was tested and found to exert a similar effect. There was striking variation in the degree of inhibition produced by different samples of horse blood and, of five strains of the organism examined, the T1 vaccine strain was the most susceptible. The results suggested that the effect was not due to antibody.     

Detection of antibody against Mycoplasma mycoides subsp mycoides in cattle by an enzyme-linked immunosorbent assay.

The results of an enzyme-linked immunosorbent assay (ELISA) For the detection of antibody against Mycoplasma mycoides subsp mycoides are presented. Antibody was detected in the sera of cattle at least 19 months after recovery from an infection and at least 23 months after vaccination. Almost half the sera of some animals in an area of Nigera where contagious bovine pleuropneumonia is enzootic contained antibody. Antibody was rarely detected when the same sera were examined by other established serological tests, emphasising the sensitivity of the ELISA.     

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.     

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.