Molecular detection of “Candidatus Rickettsia tarasevichiae” by Loop-mediated Isothermal Amplification (LAMP) of the ompA gene
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"Candidatus Rickettsia tarasevichiae" (CRT) is increasingly being recognized as a disease causative agent in China and poses a great challenge to public health. Rapid and accurate detection is indispensable for laboratory diagnosis of infection caused by CRT and its surveillance in ticks. In the present study, a novel DNA-based loop-mediated isothermal amplification (LAMP) assay targeting the ompA gene was developed for the detection of CRT in tick samples. A set of universal primers specific to CRT were designed using PrimerExplorer V5 software. The analytical sensitivity, evaluated using recombinant plasmids containing the ompA gene, reached up to 1 copy per reaction, greater than that of the PCR assay targeting the same gene. This LAMP assay specifically detected CRT and showed no cross-reaction with other species common in China within the genus Rickettsia. In addition, this newly developed LAMP assay presented high diagnostic sensitivity of CRT detection validated by known positive DNA samples from ticks and simulated clinical samples. The applicability of the LAMP assay was evaluated by screening CRT from ticks, and the result showed that CRT circulation in Weichang County, China, was confirmed. Our findings indicate that this LAMP method is sensitive and specific for the detection of CRT and may have a potential application in the detection of CRT infection in patients and ticks.The transfer of drug-resistant plasmid from Escherichia coli to pathogenic vibrios was studied by mixed culturing.Results showed that plasmid pBR322and pBR325could be transferred from E.coli to pathogenic vibrios.Some tested vibrio could be obtained plasmid within30min of mixed culturing.The transfer rate of plasmid increased with inˉcreasing culture time at24h the transfer rate was4.62×10 -6 ～1.18×10 -5 .Drug-resistant plasmid pBR322and pBR325were in pathogenic vibrio.In the initial stages of culture,no plasmid was lost,but as the culture time increased,some vibrio would lose plasmid and became sensitive to antibiotic.72h after culture,plasmids pBR322and pBR325lost in V.parahaemolyticus were10%and9%respectively,while in V.alginolyticus,22%and19%was lost.Bacterial drug-resistance level increased significantly after plasmid was obtained,but variety was seen between different bacteria indicating the expression level of drug-resistant plasmid varied.
Vibrio alginolyticus
Pathogenic Escherichia coli
Pathogenic bacteria
Plasmid preparation
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Transferability
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Phytoplasma
Coding region
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Ampicillin resistance in Haemophilus influenzae and Neisseria gonorrhoeae is most commonly due to plasmid-mediated production of the TEM β-lactamase. The H injluenzae plasmids may have evolved by insertion of various antibiotic resistance transposons into a phenotypically cryptic plasmid found in one of 699 isolates of H. influenzae examined. The small, nonconjugative, β-lactamase-specifying plasmids of N. gonorrhoeae and Haemophilus species are highly related. Phenotypically cryptic plasm ids found in several epidemiologically distinct isolates of Haemophilus parainfluenzae are highly related to the β-lactamase plasmids but carry no transposon A (TnA) sequences. This evidence strongly favors the hypothesis that the β-lactamase plasmids evolved by the insertion of TnA (possibly introduced from enteric bacteria) into cryptic plasmids resident in H. parainjluenzae.
Neisseria gonorrhoeae
Haemophilus parainfluenzae
Amp resistance
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The presence of a single apparently cryptic plasmid of approximately 36 MDa was demonstrated in the virulent Dodge strain of Legionella pneumophila. 'Tagging' of the plasmid with Tn5 enabled transfer to be demonstrated to other strains of Legionella (though not to Escherichia coli or Pseudomonas aeruginosa) as well as a definitive assessment to be made of its stability. Plasmid carriage confers resistance to UV light probably by means of an error-prone UV repair system. The plasmid is compatible with plasmids of the IncP and IncW incompatibility groups.
Legionella
Strain (injury)
Legionnaires' disease
Ultraviolet light
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Arthropods commonly carry maternally inherited intracellular bacterial symbionts that may profoundly influence host biology and evolution. The intracellular symbiont Rickettsia sp. nr. bellii swept rapidly into populations of the sweetpotato whitefly Bemisia tabaci in the south-western USA. Previous laboratory experiments showed female-bias and fitness benefits were associated with Rickettsia infection, potentially explaining the high frequencies of infection observed in field populations, but the effects varied with whitefly genetic line. Here, we explored whether host extranuclear or nuclear genes influenced the variation in the Rickettsia-host phenotype in two genetic lines of the whitefly host, each with Rickettsia-infected and uninfected sublines. Introgression between the Rickettsia-infected subline of one genetic line and the Rickettsia-uninfected subline of the other was used to create two new sublines, each with the maternally inherited extranuclear genetic lineages of one line (Rickettsia, two other symbionts and the mitochondria) and the nuclear genotype of the other. Performance assays comparing the original and new lines showed that in addition to Rickettsia, the interaction of Rickettsia infection with host nuclear genotype influenced development time and the sex ratio of the progeny, whereas the extranuclear genotype did not. Host nuclear genotype, but not extranuclear genotype, also influenced the titre of Rickettsia. Our results support the hypothesis that differences in host nuclear genotype alone may explain considerable within-population variation in host-symbiont phenotype and may contribute to the observed variation in Rickettsia-whitefly interactions worldwide.
Nuclear gene
Whitefly
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We have found an avian pathogenic Escherichia coli (APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli (UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. coli strain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coli and their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.
Pathogenic Escherichia coli
Strain (injury)
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The effects of large plasmids on different virulence characteristics of Proteus mirabilis strains mostly from clinical origin were studied. Moreover the inhibitory effect of urea and its derivatives on the swarming property of the strains was investigated. A. All strains were screened for plasmid detection, antibiotic resistance and swarming ability. B. Three multiresistant plasmid-carrying strains (PM5, P49 and P991) were cured and two transconjugants (G9pPM5 and G9pP49) were obtained by conjugation between two p+ donors (PM5 and P49) and one p- recipient (G9). C. By comparing the virulence properties of cured and transconjugant strains with their parental isolates it was found that; 1. Plasmids confer resistance to P. mirabilis strains against one or more antibiotics. 2. The presence of most plasmids reduces the swarming ability of the strains. 3. Plasmids affect the motility and flagellation of P. mirabilis strains. 4. Plasmids enhance the adherence property of their host strains to inert surfaces and uroepithelial cells as well as autoagglutination. 5. Plasmids increase the hydrophobicity of P. mirabilis strains. 6. The presence of plasmids reduced the growth rate of the strains. This effect was more apparent in iron-restricted medium. 7. Plasmids reduced the growth rate of their host strains in the presence of detergent (SDS). 8. The presence of plasmids reduced the survival of P. mirabilis strains in human and rabbit serum. 9. Plasmids decreased the survival of the strains in aquatic systems. 10. Plasmids reduced the production of urease and increased some others such as haemolysin and protease. D. Urea and some of its relatives inhibited the swarming property of P. mirabilis strains and this effect was concentration dependent.
Swarming (honey bee)
Swarming motility
Hemolysin
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Bovine viral diarrhea virus (BVDV) is a pathogen that infects cattle, and is globally important. It causes substantial financial losses to the livestock industry. In the current study, a one-step reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay was set up for rapid and efficient detection of BVDV. For this purpose, four primers were designed to recognize six distinct regions on the target RNA based on a highly conserved sequence in the 5΄ UTR of the BVDV genome. Eighty blood specimens were collected from bovines suspected to suffer from BVDV infection, and were tested in parallel by RT-LAMP and RT-PCR. Twenty four of these samples were positive by RT-LAMP, while twenty were positive by RT-PCR. The RT-LAMP detection limit was estimated to be approximately 70PFU /mL of virus. Comparison of RT-PCR with RT-LAMP in this study revealed the recent developed RT-LAMP a highly sensitive and specific for BVD virus detection in the clinical samples.
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