Ticks are notorious vectors for various pathogens that cause infections in animals and humans worldwide. Rickettsia spp., a zoonotic tick-borne pathogen that could be used as a weapon agent, is widely spread in China. In the present study, ticks were collected for species identification and Rickettsia screening. PCR amplification targeting the tick 18s rRNA gene was first conducted for species validation, and then, amplification was conducted for the Rickettsia housekeeping gene for the infection rate and phylogenetic analysis. The collected ticks were identified as Haemaphysalis longicornis , 7.36% of which were Rickettsia -positive. The phylogenetic analysis showed that the Rickettsia in the parasitic ticks belonged to a novel genotype, whose closest genetic relationship was with Rickettsia heilongjiangenesis . The samples were collected in Dandong, a city on the border between China and North Korea. Considering the geographical and biological situations of the sampling sites, more extensive surveillance and risk evaluation of the tick species and tick-borne diseases are required.

BackgroundBrucella is an important pathogen causing Brucellosis. Vaccine strains obtained by a single knockout cannot combine low virulence and immunogenicity. Our study modified the SD sequence and spacer sequence of the RBS of Brucella to affect its protein expression. We altered the RBS of LPS-associated genes to reduce LPS-associated protein expression while retaining LPS integrity.ResultsWe first established an evaluation system based on the reporter gene red fluorescent protein mCherry. The mCherry expression could be changed by altering the Shine Dalgarno sequence and spacer sequence of RBS. After optimizing the Shine Dalgarno sequence, mCherry expression was increased 4-fold in E. coli and decreased by 1/4 in Brucella. The mCherry expression was increased 1.5-fold in E. coli and decreased to 1/2 in Brucella when the length of the spacer sequence was 0. When the spacer sequence was NA (N = 4, 8, 12nt) or NG (N = 4, 8, 12nt), mCherry expression was reduced in both E. coli and Brucella. Accordingly, two mutant strains were constructed in an attempt to decrease the expression of LptA and LpxO, Brucella LPS-related genes, by 1/4. Silver staining experiments of LPS SDS-PAGE revealed an alteration in the composition of LPS in the two mutant strains. Polymyxin B experiments revealed that both mutant strains were more sensitive to Polymyxin B resistance.Conclusion: In Brucella, the expression of the target gene could be affected by changing the length or the composition of the RBS sequence. The LPS gene remained unchanged while reducing the expression of its associated protein, achieving the original goal of reducing bacterial virulence while retaining immunogenicity. It is a promising strategy to improve the safety and efficacy of vaccines.

Ticks are vectors for many infectious diseases, such as spotted fever group (SFG) rickettsioses and borreliosis, and are valuable in the study of pathogen ecology. Ticks have several growth stages that vary considerably in size; therefore, in most cases, DNA extracted from ticks is insufficient for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased amplification of DNA from tick samples before analysis is a major requirement for subsequent ecological surveys and other studies. Phi29 DNA polymerase, an enzyme that exhibits strand displacement activity, can exponentially amplify DNA randomly, generating large quantities of DNA. In the present study, we developed a Phi29-based unbiased exponential amplification (PEA) assay to obtain sufficient tick DNA for genetic analysis. By using tick-borne pathogen detection and genotyping as a model, we tested and evaluated the feasibility of the assay. DNA was extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 105 fold. The amplified products were successfully used for pathogen screening and genotyping. Rickettsia was successfully detected and genotyped in samples with amplified DNA from single ticks. Furthermore, we identified a new genotype of Rickettsia from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can be extended to other applications where the quantity of DNA is greatly limited.

Ticks are vectors for many infectious diseases such as spotted fever group (SFG) rickettsioses and borrelioses. Ticks are valuable material for pathogen ecology study. Ticks have several growth stages with significant varying size, and therefore, in most cases, the collected ticks cannot provide sufficient DNA for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased pretreatment of the tick samples for subsequent analysis is an urgent need for subsequent ecological survey and other studies. Phi29 DNA polymerase, an enzyme with strand displacement activity, could exponentially amplify DNA randomly and non-biasedly, generating large quantities of DNA. In the present study, we developed a Phi29 based unbiased exponential amplification (PEA) assay for unbiased treatment of sample nucleic acid to provide sufficient DNA for genetic analysis. By using tick borne pathogen detection and genotype as a model, we tested and evaluated the feasibility of the assay. Nucleic acid were extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 10 5 folds. The amplified products were successfully used for pathogen screening and genotyping. With the amplified DNA from single tick, Rickettsia was successfully detected and genotyped. A new genotype of Rickettsia was identified from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can also be extended to other applications where samples are greatly limited.