Study On Aerobiological Characteristics Of Yersinia Pestis And Its Pathogenicity On Mice Infected By Intratracheal Inoculation

Plague,an ancient and severe infectious disease,is still prevalent in some parts of the world.Y.pestis is the causative pathogen of plague,and its natural host is wild rodents.It can be transmitted to animals and humans by infected fleas.Plague can come in different disease forms,such as pneumonic,bubonic,or septicemic plague,among which pneumonic plague has the highest mortality rate?100% of deaths without proper treatment?.Although many studies on Y.pestis have been reported,little is known on the air-borne and pathogenic processes of the most lethal clinical type--pneumonic plague.Y.pestis spreads through the air in the form of aerosols,then inhaled into the body to cause pneumonic plague.In order to prevent and control the disease,it is of great importance for studying the characteristics of the aerosol of Y.pestis.In this study,we conducted a systematical study on the in vitro biological characteristics,air biological characteristics and pathogenicity of Y.pestis 201 strain and caf1 deletion strain in liquid aerosol forms,and established a mice model of Y.pestis infection by aerosolized intratracheal inoculation?i.t.?.This study will provide an insight into the air transmission and infection mechanism of Y.pestis aerosols.Firstly,we studied the aerobiological characteristics of Y.pestis liquid aerosol with the appropriate generators,generators and sampling fluids.In this study,we compared the occurrence and sampling differences of Y.pestis that generated by six kinds of sampling fluids?saline,PBS,BHI culture fluids,the above three sampling fluids added 0.01% Tween80 + 0.005% Olive oil respectively?,four kinds of biogenic liquids?saline,PBS,the above two biogenic liquids were added with 0.05% poloxamer respectively?,and two kinds of samplers?Collison 6jet generator and sieve plate vibration generator?.It was found that PBS was more suitable as a sampling solution in a short time?5 min?;0.01% Tween 80 + 0.005% olive oil has a protective effect on bacterial activity during a long sampling process?30 min?;saline is more suitable for spraying solution;Collison 6jet generator and sieve plate vibration aerosol generator are both suitable for the aerosol generation of this bacteria,but the latter cannot occur for more than 15 min.Aerosols of Y.pestis 201 strains were artificially produced in the airtight cabin of aerosol microenvironment.Sodium fluorescein indicator was added into the occurring liquid of some groups.The aerosols in the cabin were monitored or sampled by aerodynamic particle size spectrometer,Anderson six-stage sampler and Biosampler sampler at different time points,and then the sampling dishes were counted for live Y.pestis culture.The samples were detected by qPCR for Y.pestis gene copy number and by spectrophotometry for sodium fluorescein concentration.Then the aerodynamic particle size spectrum,sedimentation and decay of Y.pestis liquid aerosol were comprehensively analyzed.The results showed that the median diameter of aerodynamic mass?MMAD?of Y.pestis aerosol total particles was 2.35 ?m.The count median aerodynamic diameter?CMAD?of the number of biological particles was 2.8 ?m,the settling time of 90% total particles was 607 min,and the decay time of 90% biological particles(T₉₀)was 2.5 min.The above results showed that the liquid aerosol of Y.pestis could be inhaled into the lower respiratory tract or alveoli by human body,and could remain stable in the confined space for several hours,but would lose its infectivity within a few minutes.Subsequently,to understand the pathological changes and immune response of mice that infected with Y.Pestis by aerosolized i.t.,we monitored the clinical symptoms of the mice and determined the bacterial load,acute reaction time protein,cytokines and histopathological changes in mice after 100 times LD₅₀ of Y.Pestis challenge.The result of survival showed that all the mice in i.t.infected group died within 3 to 6 days post-infection?p.i.?.The bacteria in the lung were detected on 1h p.i.,the bacteria in the spleen were detected on 24 h p.i.,and the bacteria in blood were detected on 48 h p.i..The results of pathological examination showed that the pulmonary hemorrhage and edema were observed in the lung,and splenic inflammatory necrosis were observed in spleen at 72 h after challenge.The results of cytokine test showed that,the levels of C-reactive protein and penetration in the lung homogenate and serum of mice in the infected group increased.Overall,the results indicated that after infected via i.t.,Y.Pestis proliferated robustly in mice,caused pathological changes in lungs and spleens.On the other hand,within 72 h after Y.pestis infection,the body will systematically recruit inflammatory cells and release inflammatory factors,to produce humoral immunity and cellular immunity to resist bacterial infection.Finally,to explore the effect of the caf1 gene deletion on auto coacervation,air decay and virulence of Y.Pestis in mice,we constructed a caf1 deletion strain of Y.pestis using CRISPR/Cas12 a based gene editing method.The deletion of caf1 was confirmed by PCR.The in-vitro biological characteristics?including morphology,growth rate,aggregation rate?,aerobiological characteristics and virulence of caf1 deletion mutant strain were determined and compared with those wild type 201 strain.PCR results showed that caf1 deletion mutant strain was successfully constructed.The morphology and growth rate of the mutant strain did not change significantly compared with those of the wild strain.Compared with the wild strain,the caf1 deletion mutant strain can gather and deposit from the liquid medium more quickly.LD₅₀ analysis showed that the lethal dose of caf1 deletion mutant strain to mice infected via aerosol route reduced significantly.Furthermore,the virulence of caf1 deletion mutant strain to mice were significantly reduced compared with the wild type strain infected via intratracheal inoculation,nasal drops,subcutaneous and tail vein injections routes.The results showed that the deletion of caf1 gene enhance the aggregation rate of Y.pestis,but reduce the virulence of Y.pestis to mice,and did not change its decay time in air.In summary,we investigated the air biological characteristics of Y.pestis liquid aerosols,the pathogenicity of Y.pestis liquid aerosols in mice.We also compared the in vitro biological characteristics,aerobiological characteristics and animal pathogenicity between Y.pestis 201 strains and the caf1 gene deletion mutant.This study will provide theoretical support and experimental basis for the prevention and control of plague.