High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.     

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.     

Analysis of the life cycle of Mycoplasma gallisepticum

Morowitz, Harold J. (Yale University, New Haven, Conn.), and Jack Maniloff. Analysis of the life cycle of Mycoplasma gallisepticum. J. Bacteriol. 91:1638-1644. 1966.-A series of electron microscope observations on Mycoplasma gallisepticum strain A5969 have been made by use of thin-section techniques and negative staining. The methods presented a consistent picture of a postdivision cell, which contains a fibrillar nuclear region, surrounding ribosomal region, highly organized bleb at one end of the cell, granular infrableb region, and bounding unit membrane. Cell division commenced with the appearance of a second infrableb area at the end of the cell opposite the original bleb. A new bleb grew in this area, and the cell then elongated. The nuclear material segregated into two parts separated by a band of ribosomes. A constriction appeared, in this central ribosome-packed area, leading to the formation of two daughter cells. The following was noted: the cells were very small (volume, 5 x 10(-14) cm(3)); each cell was highly structured and strongly ordered; and the replication appeared to be a very precisely programmed series of events.     

Mixed Infection with cagA-Positive and cagA-Negative Strains of Helicobacter pylori

Background Helicobacter pylori infection has been implicated strongly in the pathogenesis of gastritis, peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma, but the reasons for these widely different clinical outcomes are unknown. The aim of this study was to determine whether these differences could be due in part to mixed infection in the same individual, with bacteria having differences in pathogenic factors associated with ulcers.
Materials and Methods. The cagA gene of H. pylori was used to test for mixed infection because it is present in only some strains, and its presence has been associated with ulcers. Polymerase chain reaction (PCR) assays for the cagA gene were applied to H. pylori culture isolates and endoscopic gastric aspirates. Individual bacterial clones were tested for genetic similarity by random primer amplification and restriction endonuclease digestion of urease gene PCR products.
Results. The majority of H. pylori -positive patients had strongly cagA -positive culture isolates and endoscopic samples (62.5% and 69.6%, respectively). However, many of these patients had evidence of mixed infection with cagA negative and cagA positive strais in cultures isolates and endoscopic samples (25% and 17.4%, respectively). Mixed infection was found to be due to genetically unrelated strains in two patients in whom genetic analysis was performed.
Conclusion. Mixed infection with differences in substrain pathogenic factors might occur in H. pylori infection and might contribute to differences in clinical outcome.     

兰州地区实验动物肺支原体感染调查和分离鉴定

目的了解兰州地区啮齿类实验动物的肺支原体感染情况和感染菌株.方法用分离培养法对兰州地区640只啮齿类实验动物肺支原体的感染情况分春夏秋冬进行调查,并对分离株进行克隆纯化,从形态学,生化特性和血清学方面进行鉴定.结果肺支原体在普通级小鼠中的感染率为23%,普通级豚鼠、地鼠、大鼠和清洁级小鼠未发现有感染,且感染率与季节无明显相关性.分离株经鉴定均为支原体科支原体属肺支原体.     

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.     

鸡毒支原体强毒株与F疫苗株双重PCR检测方法的建立

目的建立能同时检测MG强毒株和F疫苗株的双重PCR技术。方法根据鸡毒支原体(MG)R株的PvpA基因序列和F疫苗株假定的α磷酸海藻糖酶基因序列,设计2对引物R1、R2和F1、F2,在单一PCR的基础上,建立检测MG强毒株和F疫苗株的双重PCR方法,并运用该双重PCR方法对临床样品进行检测。结果在330 bp和444 bp处分别出现预期的特异性DNA扩增条带,敏感性试验显示该体系能检测出0.45 ng的MG R株DNA和0.25 ng的MG F疫苗株DNA。临床样品MG强毒株阳性检出率为79.69%,高于常规分离培养鉴定法。结论成功建立检测两种毒株的双重PCR技术,为根除鸡群中MG野毒株、建立无MG的阴性鸡群提供新的技术手段。     

Evidence for Complementation of Plant Potyvirus Pathogenic Strains in Mixed Infection

Several strains of soybean mosaic virus (SMV) can be differentiated on the basis of the phenotypic response of various soybean cultivars (e.g., the soybean line Williams ‘82 is susceptible to all SMV strains, whereas the lines P. I. 96983, L78-379, and Davis are functionally immune to SMV strain G2 but susceptible to strains G7 and G7a). Inoculation of the immune lines with G2, followed 2 days later by inoculation with G7 or G7a, resulted in systemic spread of the avirulent SMV G2. Further evidence, suggests that complementation groups of SMV strains may exist.     

New Method for the Isolation of Membranes from Mycoplasma gallisepticum

Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH 9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greater than a cell suspension at the same protein concentration in buffered saline. Membranes prepared from cells lysed by this method at pH 10 were relatively free from cytoplasmic contaminants as shown by electron microscopy of thin sections. The membranes retained their hemagglutination activity, gave reactions in immunodiffusion tests identical to those obtained by osmotic lysis and sonic treatment, and showed a similar pattern of protein bands by polyacrylamide disk electrophoresis. When inoculated into rabbits, the membranes gave rise to antibodies active in growth-, metabolic- and hemagglutination-inhibition tests. On the average, membranes obtained by lysis at pH 10 contained 44% of the original cell protein. The method is simple, giving high yields of membranes, and may be adaptable to other mycoplasmas.     

Heterogeneity Among Strains of Mycoplasma granularum and Identification of Mycoplasma hyosynoviae, sp. n

Twelve filtrable, pleomorphic organisms isolated from swine joints and respiratory tracts had typical colonial and microscopic characteristics of mycoplasmas. They resisted penicillin and did not revert to cell wall-producing bacterial forms in media devoid of bacterial inhibitors. The morphological and growth characteristics of these mycoplasmas were similar to those described previously for Mycoplasma granularum. However, a new name, M. hyosynoviae, is proposed for them since they differed biologically, serologically, and electrophoretically from the prototype strain of M. granularum. M. hyosynoviae required sterols, was stimulated by gastric mucin, and metabolized arginine; however, it did not metabolize urea, ferment glucose, or reduce tetrazolium. The organism produced "film and spots" on horse serum-supplemented medium and produced alpha hemolysis of guinea pig and sheep erythrocytes; however, it did not digest serum, produce phosphatase, or hemadsorb guinea pig or swine erythrocytes. M. hyosynoviae was distinguished from three other swine mycoplasmas, M. granularum, M. hyorhinis, and M. laidlawii, by means of acrylamide gel electrophoresis, growth inhibition, metabolic inhibition, and immunodiffusion techniques. It was also serologically and electrophoretically distinct from 13 additional non-swine mycoplasmas which require sterols and metabolize arginine.     

Interactions between Viral and Prokaryotic Pathogens in a Mixed Infection with Cardiovirus and Mycoplasma

In the natural environment, animal and plant viruses often share ecological niches with microorganisms, but the interactions between these pathogens, although potentially having important implications, are poorly investigated. The present report demonstrates, in a model system, profound mutual effects of mycoplasma and cardioviruses in animal cell cultures. In contrast to mycoplasma-free cells, cultures contaminated with Mycoplasma hyorhinis responded to infection with encephalomyocarditis virus (EMCV), a picornavirus, but not with poliovirus (also a picornavirus), with a strong activation of a DNase(s), as evidenced by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) immunofluorescence assay and electrophoretic analysis of host DNA. This degradation was reminiscent of that observed upon apoptosis but was caspase independent, judging by the failure of the specific pan-caspase inhibitor Q-VD-OPh to prevent it. The electrophoretic mobility of the enzyme responsible for DNA degradation and dependence of its activity on ionic conditions strongly suggested that it was represented by a DNase(s) of mycoplasma origin. In cells not infected with EMCV, the relevant DNase was dormant. The possibility is discussed that activation of the mycoplasma DNase might be linked to a relatively early increase in permeability of plasma membrane of the infected cells caused by EMCV. This type of unanticipated virus-mycoplasma “cooperation” may exemplify the complexity of pathogen-host interactions under conditions when viruses and microorganisms are infecting the same host. In the course of the present study, it was also demonstrated that pan-caspase inhibitor zVAD(OMe).fmk strongly suppressed cardiovirus polyprotein processing, illustrating an additional pitfall in investigations of viral effects on the apoptotic system of host cells.Traditionally, virologists are predominantly studying properties of individual viruses and much rarely interactions between different viruses. Even less attention is paid to interactions between viruses and microbes, although animal and plant viruses quite often share, in the natural environment, their ecological niches with microorganisms. Interactions of microbes with viruses other than phages are essentially a virgin soil of either virology or microbiology. The scarcity of relevant information may be illustrated by a recent review focused on the interaction between enteric viruses and enteric bacteria (23). There is ground to believe, however, that the impact of microbial flora on viral growth and pathogenicity (and vice versa) is much broader and deeper than is currently thought. One of the reasons for such a belief is the well-known effect of bacteria (and of their metabolic products) on diverse signaling systems of animal cells, in particular those involved in innate immunity (14, 41, 85, 89). A recent relevant example is demonstration that a prokaryotic infection may protect drosophila from a variety of RNA viruses (39). On the other hand, viral infection may significantly alter the outcome of concomitant bacterial infections. For example, it was reported that herpesvirus latency in mice may confer significant resistance to Listeria and Yersinia pathogens (8).The aim of the present report is to demonstrate, in a model system, profound mutual effects of mycoplasma and cardioviruses during their interaction with animal cells. Cardioviruses, a genus in the picornavirus family, are represented by such widely investigated viruses as encephalomyocarditis virus (EMCV) and its strain mengovirus (MV), as well as Theiler''s murine encephalomyelitis virus (TMEV). Cardioviruses are pathogens naturally affecting many animal species, in particular, rodents, pigs, and some primates (49). Recently, TMEV-related viruses have been implicated in human pathology (1, 12, 21, 29, 45, 46, 51, 96). Picornaviruses, which also include such viruses as poliovirus, hepatitis A virus, foot-and-mouth disease virus, and many others, possess a 7.2-8 kb-long single-stranded RNA genome of positive polarity and share general features of their reproduction mechanism (2). The genomic RNA contains a large single reading frame (certain strains of TMEV, but not EMCV, encode also a protein, L*, in an alternative frame; see reference 47) translated into a polyprotein, which is processed into a dozen “mature” proteins by a series of proteolytic events.Interaction of picornaviruses with susceptible cells may have different outcomes: it may result in cytopathic (necrotic) effect (CPE) or apoptotic death, or it may lead to persistent infection. The character of these outcomes has obvious implications for the pathogenesis of the viral disease. Necrotic death is a strong trigger of inflammatory reactions. Apoptosis, which leads to degradation of host DNA and fragmentation of cells into membrane-coated so-called apoptotic bodies and their eventual “consumption” by macrophages and other scavenger cells, could limit not only the spread of the viral progeny but also prevent potential damages to the neighboring cells caused by inflammation. Therefore, apoptosis is generally considered to represent a defensive host reaction. The fate of the picornavirus-infected cell depends on the genetic properties of the both partners of virus-host interaction, as well as on the status of cell differentiation and environmental conditions (3, 13, 69, 70, 79, 80, 86). In particular, cardioviruses may evoke apoptotic reaction in certain, especially partially restrictive, cells (42, 43, 75, 76, 92).Mycoplasmas are the smallest prokaryotic obligate parasites devoid of cell wall and of many synthetic capabilities and able to trigger a variety of diseases, such as pneumonia, uretritis and many others; they also frequently contaminate cultured cells. Mycoplasmas could be attached to plasma membrane of eukaryotic cells but in some cases they are able to invade the cells as well. They may exert a variety of effects on signaling systems of animal cells affecting, in particular, their innate immunity (67, 71).We will demonstrate here that, in several mycoplasma-contaminated cell cultures, infection with EMCV (but not with poliovirus) results in activation of the microorganism-derived DNase(s), which accomplishes degradation of host cell DNA resembling that occurring during apoptosis but which was caspase independent. The same but mycoplasma-free cells respond to EMCV infection with a canonical CPE (70). This type of unanticipated virus-mycoplasma “cooperation” may illustrate the complexity of pathogen-host interactions under conditions when both viruses and microorganisms are present.     

Evaluation of Some Serological Techniques for The Identification of Mycoplasma Hyorhinis and Mycoplasma Suipneumoniae

Eighteen strains of Mycoplasma hyorhinis and a strain of Mycoplasma suipneumoniae were tested in 4 serological tests, i. e., disc growth inhibition, metabolic inhibition, indirect haemagglutination and indirect epi-immunofluorescence. Only with immunofluorescence could all tested strains of M. hyorhinis be shown; no cross-reactions between M. hyorhinis and M. suipneumoniae could be detected. The other tests failed in many cases to identify strains of the same species, and they gave cross-reactions between M. hyorhinis and M. suipneumoniae.     

A comparison of the methods of the European Pharmacopoeia for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae

The methods specified in the European Pharmacopoeia for the detection of Mycoplasma synoviae and Mycoplasma gallisepticum as contaminants of vaccines were compared by investigating the serological responses of chicks inoculated with dilutions of mycoplasma cultures, these cultures being titrated in parallel in vitro. Inoculation by the intrathoracic route proved to be as sensitive as, or more sensitive than the other methods and was of similar sensitivity to the in vitro titrations for both agents.     

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.     

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100–120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.

     

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum.

The membrane-bound ATPase of Mycoplasma gallisepticum selectively hydrolyzed purine nucleoside triphosphates and dATP. ADP, although not a substrate, inhibited ATP hydrolysis. The enzyme exhibited a pH optimum of 7.0 to 7.5 and an obligatory requirement for divalent cations. Dicyclohexylcarbodiimide at a concentration of 1 mM inhibited 95% of the ATPase activity at 37 degrees C, with 50% inhibition occurring at 22 microM dicyclohexylcarbodiimide. Sodium or potassium (or both) failed to stimulate activity by greater than 37%. Azide (2.6 mM), diethylstilbestrol (100 micrograms/ml), p-chloromercuribenzoate (1 mM), and vanadate (50 microM) inhibited 50, 91, 89, and 60%, respectively. The ATPase activity could not be removed from the membrane without detergent solubilization. Although most detergents inactivated the enzyme, the dipolar ionic detergent N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (0.1%) solubilized approximately 70% of the enzyme with only a minor loss in activity. The extraction led to a twofold increase in specific activity and retention of inhibition by dicyclohexylcarbodiimide and ADP. Glycerol greatly increased the stability of the solubilized enzyme. The properties of the membrane-bound ATPase are not consistent with any known ATPase. We postulate that the ATPase functions as an electrogenic proton pump.     

Immunoperoxidase technique for identification of Mycoplasma gallisepticum and M. synoviae.

The direct immunoperoxidase technique was applied to the identification of Mycoplasma gallisepticum and M. synoviae by staining colonies on the agar plate. The results of this technique applied to 50 isolates of M. gallisepticum and M. synoviae correlated with those of the agar gel precipitation test to the same isolates. The immunoperoxidase technique was proved to be a specific and reliable method for the identification of M. gallisepticum and M. synoviae.