Isolation,Identification And Pathogenicity Of Tick-Derived Bacillus In Mice

Bacillus are widely distributed in nature such as insects,wildlife,soil,air,plant leaf surfaces and sewage.Bacillus species has great use value in agriculture and animal husbandry.Recently,Bacillus are widely studied as biological control agents,because they can inhibit many pathogens.Among them,Bacillus thuringiensis is the most popular microbial insecticide and even environment friendly biological pesticides at home and abroad.It can produce intracellular protein crystals which is toxic to a large number of insects and their larvae but nontoxic to human and animals.In this experiment,ticks with the freedom of movement were collected in Hulun Buir area with the white cloth flag method.Fresh individual tick was selected for microbial diversity analysis,and on the basic knowledge of the distribution of tick-derived Bacillus,and the traditional bacterial culture method was used for bacterial isolation,culture and identification.Single bacterial colonies were used for Gram staining and morphology observation and transferred to 5%sheep blood agar plates for haemolysis.Pure cultures were used for biochemical experiments and 16S r RNA sequence genetic evolution analysis.Then the result showed that,the tick species type collected in this experiment was grassland ticks,one of the dominant ticks in Hulunbuir region.Four gram-positive bacillus strains were isolated from 898 ticks tissue sample:Bacillus proteolysin,Bacillus parasides,Bacillus thuringiensis and Bacillus cereus,IMH/B-1 strain,IMH/P-1 strain,IMH/T-1 strain and IMH/C-1 strain,respectively;chitinase genes of B.thuringiensis and B.proteolysin were detected by PCR method,but it was not detected in Bacillus cereus and Bacillus parascarides;B.parascari and B.proteolysin were selected for mouse pathogenicity testing,with the results showing that all the mice with 1.49×10¹⁰cfu/m L injection died within 24 hours,displaying acute clinical symptoms due to virulent strains,with the characteristic pathological changes mainly concentrated in the liver,kidney,spleen,intestine and brain tissues.Through the isolation,identification and safety evaluation of four kinds of Bacillus species,this experiment laid a foundation for the future application of biologically active substances such as chitinases of bacillus or britinases,and accumulated basic data for the next biological control research on ticks.