Development Of A Nose-only Bioaerosol Exposure System For Small And Medium Size Animals And Study On T He Pat Hogenicity Of Yersinia Pestis Infection Via Aerosolized Nose-only Exposure

During the period from the end of 2019 till the start of 2020,the new corona virus had broken out in China and spread globally^[1].As one of the virus potential spreading pathways,aerosol had attracted extensive attention and numerous analysis had been initiated by following this potential pathway.With the intention of successfully analyzing the infection caused by pathogen aerosol pathogens and its relative protecting methods,consistent and reliable animal models infected through inhalation play essential roles.According to the FDA relative guidelines,it is suggested that the infected animal models should be established under the estimated environmental exposure in which human have experienced^[2,3].Due to non-anesthesia requirement and the similarity of inhalational mechanism between human and animals,nose-only exposure is normally selected as the first breakthrough point for this type of analysis.However,to establish the animal models which have nose-only exposure requires large scale aerosol Nose-only exposure devices which are development under professional technology.European countries impose strict technology protection and embargo on this type of devices,along with insufficient China domestic self-developing devices,China domestic inhaled pathogen infection analysis are carried out by applying Intranasal/Intratracheal Instillation instead,which results in aerosol nose-only animal model analysis lagging behind,especially when facing high violent inhaling pathogen analysis which requires high biological protection.This analysis is aiming to develop the new generation of animal aerosolnose-only exposure system which can be utilized in the laboratory which requires high level biological protection.Based on this system,the Yersinia pestis was selected as type strain because of its pathogenic violence and aerosol spreadable characters.Through understand the nose-only aerosol spreading methods to establish aerosol animal nose-only exposure models,the character of the aerosol infection can be analyzed.The analysis will consummate the platform of animal aerosol inhaled infection exposure devices technology.A new generation of Nose-only Aerosol Exposure System for small and medium size animals（NOBES）consists of four functional modules:aerosol nebulization,aerosol exposure,Class III biosafety cabinet and parameter control monitoring.The integration of the system shows unique innovative advantages:a group of design specifications of fixed cylinders meet the needs of various types of animals;the self-developed vortex scattering diluents greatly improve the uniformity and stability of aerosol transport;the installation of a variety of environmental and engineering parameter sensors improves the level of automation,the development of control software ensures high aerosol control precision;the design of the exhaust line of the transfer compartment enhances the biological safety protection;the unique transfer door design realizes good effect.After the integration of the system,a series of performance verification tests were carried out,The results showed that the median aerodynamic mass diameter of liquid aerosol of NOBES was 1.24±0.04μm、2.77±0.15μm,respectively 2.50±0.41μm,within the particle size range of aerosols that can enter the deep respiratory tract or alveoli（<5μm）;The concentration coefficient（CV）of concentration distribution of glucose aerosol and mucin sire aerosol among the three tiers of exposure tower in NOBES,the CV between the suction ports of each tier,the CV between the angles of each tier,the CV for a continuous period of 1 h and the CV for different days are all meet the requirements of the Organization for International Economic Cooperation and Development guidelines that an average concentration coefficient of variation should not exceed 20 percent^[4].Meanwhile,the uniformity of glucose aerosol in each tier（4.42%）,the repeatability（0.68%）during different days,and the uniformity of bacteria aerosol in each tier（up to 15.12%）of NOBES are better than that of other similar products,indicating that the stability of aerosol transport uniformity of NOBES reaches the international leading level.The overall filtration efficiency test>99.999%,which meets domestic and international standards,indicating that the system has good sealing property.There was no abnormal behavior in the system in the test for exposure to the nose and mouth by loading mice into a fixed tube,and The operation of nasal exposure is well adapted.To sum up,it can be inferred that the aerosol transmission performance,safety protection level,automation level and ergonomics efficiency of the new generation of mouth and nose exposure system have been comprehensively improved.Furthermore,a NOBES standard operating procedure is developed,that lays the foundation for the subsequent study of oral and nasal exposure of pathogenic aerosol animals.Using the newly developed NOBES system,we subsequently carried out research on the occurrence and transmission of Yersinia pestis liquid aerosol and the characteristics of mice inhalation.First,the liquid shock sampler cascade sampling shock test of Yersinia pestis and Serratia marcescens,aerosol generation and cascade sampling shock test were carried out.During the sampling process,the biological activity damage rate relative to Serratia marcescens was 160.36±11.46%and116.62±9.61%,indicating that Yersinia pestis is poorly tolerated.Therefore,the aerosolization and sampling techniques for this bacteria need to be studied and optimized.In addition,it was also found in the test that the liquid shock sampling has aerosol escape phenomenon.It is suggested that when testing the bacterial aerosol concentration of the inhalation port of the NOBES system by the liquid shock sampling method,the impact of biological activity damage rate of the bacterial sampling and the escape rate must be taked into consideration to calibrate the value of the aerosol concentration;secondly,a comparative study was carried out on the two aerosol generators and the two groups of transmission airflow parameters of the NOBES system,and the optimized transmission conditions of Yersinia pestis aerosol were initially obtained,that is,the Collison 12 generator was used without adding Dilution air;Next,I tested the Yersinia pestis liquid aerosol atomization factor（SF）of NOBES operating under optimized transmission conditions,and the result was 3.28×10^-7,which is better than most international counterparts^[5,6],and does not change with the date of the experiment and the concentration of the liquid,it indicates that the Yersinia pestis liquid aerosol of the NOBES system has excellent transmission efficiency and stability;Subsequently,the linear correlation and regression analysis between the concentration of the generating fluid and the aerosol concentration of the inhalation and the amount of bacterial deposition in the lungs of mice showed that both the aerosol concentration of the inhalation and the amount of bacterial deposits in the lungs of the mice were positively correlated with the concentration of the generating fluid.Corresponding linear regression equations were established to establish a method for predicting the inhalation concentration of the Yersinia pestis aerosol NOBES system and the inhaled dose in mice.Finally,the same dose of Yersinia pestis exposure through aerosol nose and aerosol lungs was compared.The distribution of bacteria in the body tissues of BALB/c mice infected by the three routes of delivery and intranasal drip for 10 minutes.It was found that the aerosol oral and nasal exposure route has the widest distribution of bacterial tissues in the respiratory system,the lowest deposition rate in the lungs,and also the particle size of the deposited particles is smaller,and the transfer of bacteria from the lungs to other organs is faster.At the same time,this pathway only infects the respiratory system and also infects the digestive tract and brain tissue meanwhile.Therefore,its animal model of inhalation infection has its uniqueness.It cannot be replaced by other means.Through the above research,we have initially grasped the characteristics of the occurrence and transmission of Yersinia pestis liquid aerosol in the NOBES system and the inhalation characteristics of mice.Then,we evaluated the characteristics of Yersinia pestis infection via oral and nasal exposure route using the optimized transmission system mentioned above.The LD₅₀ of aerosolized Yersinia pestis challenge via nasal exposure route was 155CFU（95%CI:74 CFU-283 CFU）.The mice were infected with 1.15±0.56×10⁵ CFU of aerosolized Yersinia pestis via nose-only exposure route.The results were as follows:The bacterium burden in the lung,spleen and blood of mouse increased during the infection course.The level of acute reactive protein,serum amyloid P component（SAP）,and cytokines（IFN-γ,IL-6）in mouse serum,lung homogenate and spleen homogenate also increased gradually during the infection course.The pathologic changes in the lung and spleen（presented as lung edema,lung blooding and necrosis of splenitis）were observed in mice of infected groups at 48 h or 60 h p.i.,but not in the saline inoculated group.Overall,the results above indicated that Yersinia pestis propagate and spread continuously in mice after infection via nasal exposure route,caused the pathologic lesions in the lung and spleen of mice,and induced stronger innate immune response in mice.The results above indicated that we have successfully established a mouse model of Yersinia pestis infection via nasal exposure route.In summary,this analysis solved the key practical technical issue faced by the advanced biological laboratory on nose-only aerosol exposure for small and medium size animals.The new self-developed Nose-only aerosol system for the small to medium size experimental animals improve the aerosol transferring efficiency,biological safety protection,automation and interface system.The characteristic of Yersinia pestis distribution through mice inhalation and aerosol transferability were comprehended through the succeeding tests.Yersinia pestis aerosol exposure infection model through mice Nose-only inhalation has been established by applying optimized transferring condition.This analysis provided supports from both devices and theories to respiratory pathogen infection mechanism analysis and further to vaccine and medicine evaluation.