Nucleotide sequence of Mycoplasma mycoides subspecies Mycoides plasmid pKMK1.

To facilitate the development of mycoplasmal cloning vectors, we have determined the nucleotide sequence of pKMK1, a cryptic plasmid isolated from Mycoplasma mycoides subsp. mycoides. It is 1875 bp in length and contains two open reading frames (ORFs) that share homology with ORFs from members of a large family of gram-positive bacterial plasmids which replicate via a single-stranded DNA intermediate. Putative origins of replication and candidate cloning sites have been identified.     

Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.     

Characteristics of S Organism Isolated from Methanobacillus omelianskii

Previous work showed that Methanobacillus omelianskii was a mixed culture of an ethanol-oxidizing organism called S organism and a hydrogen-utilizing methane bacterium, strain MOH. S organism grows poorly on ethanol unless a hydrogen-utilizing methanogenic bacterium is included to utilize the H(2) produced during growth. Further studies have shown that, among many substrates tested, only ethanol, n-propanol, n-butanol, isobutanol, n-pentanol, acetaldehyde, oxalacetate, and pyruvate are fermented by S organism, either alone or in combination with Methanobacterium ruminantium. It grew better in pure culture with pyruvate than with alcohols. H(2) gas phase inhibited growth on pyruvate as well as on alcohol. When grown alone on pyruvate, S organism produced mainly acetate, ethanol, and CO(2), in addition to a small amount of H(2). When combined with M. ruminantium, no H(2) and very little ethanol were produced and acetate production was increased. When M. ruminantium was present, electrons from pyruvate oxidation by S organism were channeled almost entirely to H(2) and hence to methane formation rather than ethanol. Also, S organism utilized more pyruvate when grown with M. ruminantium. Attempts to obtain better growth of S organism on ethanol by addition of many possible electron acceptors were unsuccessful. It grew best between 32 and 45 C, had a per cent guanine plus cytosine content of deoxyribonucleic acid bases of 47.27 +/- 0.1, contained no cytochrome, and could be grown on a defined medium with pyruvate as the energy and carbon source and with (NH(4))(2)SO(4) as the main nitrogen source. These and other results suggest that S organism belongs in a new genus, but assignment of a definite taxonomic status should await isolation and characterization of more strains.     

Characteristics of Mycoplasma meleagridis sp. n., Isolated from Turkeys.

Yamamoto, R. (University of California, Davis), C. H. Bigland, and H. B. Ortmayer. Characteristics of Mycoplasma meleagridis sp. n., isolated from turkeys. J. Bacteriol. 90:47-49. 1965.-A designation is proposed for a pathogenic Mycoplasma species isolated from turkeys. The organism originally was recovered from the air-sac lesion of a turkey poult in 1957, and was designated the "N" strain. Mycoplasma species with identical characteristics have since been recovered from the sinus, trachea, oviduct, vagina, semen, and bursa of Fabricius of turkeys. The organism has been recovered from many turkey flocks throughout the country. Many investigators have confirmed the original finding that this organism is antigenically distinct from other known serotypes of Mycoplasma found in poultry. The species proposed is Mycoplasma meleagridis sp. n.     

Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.     

牦牛肠道粪肠球菌的分离和鉴定

试验为获得1株与牦牛肠道共生的粪肠球菌,通过对健康牦牛粪样中的肠球菌进行分离培养、革兰染色、生化试验、16S rRNA基因测序比对等生物学鉴定,初步证实该分离菌株为粪肠球菌。此试验为进一步分析牦牛肠道菌群结构,探索牦牛耐饥、耐渴、耐粗饲、对高山草原极强适应性与其肠道菌群的关系,进一步研究与牦牛共生的粪肠球菌的生物学特性、生理特性、对宿主危害性等提供一些实验依据。     

Cancer as a Programmed Death of an Organism

The hypothesis introduces the idea that there is a critical level of mutagenesis that triggers a program of organism death by means of proliferation of killer cells. Similarly to apoptosis, which is an altruistic suicidal act of a faulty cell threatening the stability of a multicellular organism, a malignant tumor is an altruistic suicide of an individual carrier of harmful alleles threatening genetic stability of the population.     

蕈状芽孢杆菌代谢产物增强小鼠免疫力活性物质的分离、鉴定

本文的目的是对蕈状芽孢杆菌代谢产物中增强小鼠免疫力的物质进行分离、鉴定和免疫增强作用测定。在生物活性追踪的指导下, 本文通过大孔吸附树脂吸附、硅胶柱分离和葡聚糖凝胶柱纯化, 对蕈状芽孢杆菌代谢产物中增强小鼠免疫力的活性成分进行了分离, 得到了具有较强免疫增强作用的化合物(标记为M), 并对化合物M进行了波谱测定, 确定化合物M为环(脯氨酸-甘氨酸)二肽(C7H10O2N2)。以生理盐水为对照, 通过腹腔注射, 对小鼠进行了SOD活性、白细胞吞噬活性、白细胞杀菌活性3个指标测定。结果显示, 在第14天时, SOD活性和白细胞吞噬活性达到了最高值, 且同对照组相比有显著提高; 在第21天时, 白细胞杀菌活性达到了最高值, 且同对照组相比有显著提高。以上结果表明, 蕈状芽孢杆菌代谢产物中的环(脯氨酸-甘氨酸)二肽能够显著增强小鼠免疫力。     

Amoebae as Exemplary Cells: The Protean Nature of an Elementary Organism

In the nineteenth century protozoology and early cell biology intersected through the nexus of Darwin’s theory of evolution. As single-celled organisms, amoebae offered an attractive focus of study for researchers seeking evolutionary relationships between the cells of humans and other animals, and their primitive appearance made them a favourite model for the ancient ancestor of all living things. Their resemblance to human and other metazoan cells made them popular objects of study among morphologists, physiologists, and even those investigating animal behaviour. The amoeba became the exemplar of the new protoplasmic cell concept of mid-century and because its apparent simplicity made it widely generalizable it became a popular subject in a breadth of experimental investigations and theoretical speculations. It was able to do this because “the amoeba” denotes not a particular organism, but a general type of behaviour common to the cells of a range of protozoa, simple plants and higher animals. Its status as an exemplary cell also rested upon auxiliary philosophical assumptions about what constitutes a primitive characteristic and the thesis that evolution is a progressive development of order from chaos.     

Identification of a Novel Afipia Species Isolated from an Indian Flying Fox

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.     

蛋鸡J亚群白血病病毒的分离鉴定及序列分析

通过接种鸡胚成纤维细胞((CEF)及特异性单抗的间接荧光抗体反应(IFA),从中国商品代蛋鸡群中首次分离到J亚群白血病病毒(ALV-J).对其env基因编码的氨基酸序列及3'-末端(3'-Ter)序列与国内外来源于白羽肉鸡的毒株作了比较分析.结果显示,这两株病毒的gp85基因编码的氨基酸序列与国外5个毒株同源性仅为83.4%～87.3%,与国内来源于白羽肉鸡的8株病毒同源性也仅为86.4%～89.6%.gp37基因编码的氨基酸序列与5个国外毒株同源性为91.8%～97.0%,与8个国内毒株同源性为93.9%～95.9%.另外,国内来源于白羽肉鸡的各毒株的3'-Ter序列在"E"区均有明显缺失,但本次分离的来源于蛋鸡群的毒株在"E"区没有缺失突变.与所列出的13株国内外毒株相比,这两个毒株在3'-Ter的缺失最少,较接近于原型株HPRS-103.显然这两株病毒的来源不同于国内白羽肉鸡.     

Identification of Phosphocholine-Containing Glycoglycerolipids Purified from Mycoplasma fermentans-Infected Human Helper T-Cell Culture as Components of M. fermentans

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell culture (MT-4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT-4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated MT-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.     

丝状支原体丝状亚种SC型脂蛋白Q N末端基因的表达、纯化与抗原性分析

为了表达丝状支原体丝状亚种SC型(MmmSC)中国分离株HVRIⅩ脂蛋白Q(LppQ)N末端基因,将该基因经PCR扩增后克隆至原核表达载体pET32a中,经酶切、PCR、测序证实获得了重组表达质粒,转化Escherichia coliBL21(DE3)菌,经IPTG诱导后获得可溶性融合蛋白,表达量占菌体总蛋白的53.7%,用Ni-NTAHis.Bind纯化试剂盒纯化后,蛋白纯度达95%以上。表达蛋白经Western blot检测其抗原活性,结果表明纯化蛋白可与CBPP标准阳性血清发生强烈的反应,而与阴性血清不发生反应。     

Identification of the Flower-inducing Factor Isolated from Aphid Honeydew as being Salicylic Acid

Honeydew produced by the aphid Dactynotus ambrosiae when feeding on flowering or vegetative plants of the short day plant Xanthium strumarium contains an active substance capable of inducing flowering in the long day plant Lemna gibba G3. In the present study, this active material has been identified as salicylic acid through the use of gas-liquid chromatography and mass, infrared, and ultraviolet spectrometry. Authentic salicylic acid induces flowering in L. gibba G3 under strict short day conditions with an optimal response at about 5.6 μm. The possible significance of salicylic acid for the control of flowering in Xanthium or L. gibba G3 is discussed.     

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC)¹ is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe respiratory disease in cattle. It is a disease requiring official declaration to the World Organization for Animal Health (OIE) and that causes vast problems in Africa with severe socioeconomic consequences (1, 2). In 2006, 15 African countries reported 186 outbreaks of CBPP to the OIE. CBPP was eradicated from Europe in the beginning of the 20th century (3) but has reemerged in every decade since (4). Eradication was largely facilitated by slaughtering infected herds, which is still considered as the most efficient means of disease control and was successfully performed in Botswana in 1995 (5). However, this campaign was directly correlated to increased malnutrition in children (6) and is also considered to be too expensive for other African countries (2, 7). The use of chemotherapy in CBPP control is a debated subject, has long been discouraged, and is even illegal in some countries (1), mainly because of the risk of creating silent carriers of the disease (8). However, new antibiotics have shown positive effects (9), but extensive vaccinations are still considered the preferred option for prevention and control of CBPP in Africa (2, 10, 11). The vaccines currently in use are based on live attenuated M. mycoides SC strains and have several disadvantages such as short term immunity (12), poor protection as indicated in recent trials (4, 13), and even pathogenicity (13, 14).The two currently available tests for serological diagnosis of CBPP recommended by the OIE, the complement fixation test (15) and a competitive ELISA (16), are based on whole cell M. mycoides SC. For subcellular components of the organism, the genome sequence of M. mycoides SC strain PG1 (17) offers an emerging possibility to improve both diagnostic and therapeutic approaches with selected antigens. However, as for the 10 other Mycoplasma genomes sequenced, the genome sequences per se did not reveal any primary virulence factors common in other bacteria, such as adhesins or toxins (18). The few known molecular mechanisms of pathogenicity were recently reviewed (18) and include five lipoproteins studied in detail: LppA (19, 20), LppB (21), LppC (22) LppQ (23), and Vmm (24). Of these, LppQ has been used to develop an indirect ELISA (25), and Vmm, a variable surface protein, has recently been studied along with five novel putative variable surface proteins as recombinant proteins expressed in Escherichia coli (26). That study demonstrated the feasibility of producing recombinant surface proteins from M. mycoides SC in E. coli and screening for antibodies in sera from CBPP-affected bovines by Western and dot blotting.To explore further the immunogenicity of the M. mycoides SC surface proteome, a platform for multiplexed analysis of proteins using minute serum samples such as bead-based array systems (27) is desirable. One method is available from Luminex Corp. and uses spectrally distinguishable beads (28) to form an array in suspension. The array is analyzed in a flow cytometer-like instrument and can perform up to 100 simultaneous assays in a single reaction well. This platform has recently been used to determine binding specificities to antigens produced in a similar fashion (29) and to profile antibodies in serum toward six antigens of Mycobacterium tuberculosis (30).The aim of this study was to develop a rapid and highly multiplex method for affinity analysis of antibody levels in serum samples from CBPP-affected bovines against recombinant M. mycoides SC surface proteins. To facilitate this, a large set of surface proteins were cloned, expressed in E. coli, and purified. Furthermore, the bead-based assay conditions had to be optimized and verified for detection of immunoglobulin levels in bovine sera. This methodology would enable monitoring and protein-specific characterization of humoral immune responses during CBPP infections. As a secondary aim, the study was expanded to include specific IgG, IgA, and IgM responses in sera from a vaccine study with time series sampling from each animal over 8 months, covering prevaccination and 4 months postinfection.