Screening Of Swine Anti-swine Streptococcosis Resistance-related Genes

Streptococcus suis(S.suis)is an important swine and human pathogen,which not only significantly restricts the development of swine industry all over the world,but also threatens the health of human beings.Among the existing 33 serotypes(1-31,33,1/2),Streptococcus suis type 2(SS2)has the highest toxicity and is mostly widely-spread.Currently,major prevention and treatment measures of Swine Streptococcosis include management reinforcement,vaccination,and drug treatment;however,these measures have limited effectiveness.Along with the development of transgenic technology,disease-resistant breeding provides new insights to the prevention and treatment of Swine Streptococcosis.1.Susceptibility Test of Different Mice Strains to Streptococcus suis Type 2The clinical features of mice infected with SS2 are similar to those of infected swine,and different strains of mice have different susceptibilities to SS2 infection.Thus mice models can be adopted to study SS2 infection in swine.In this study,we randomly divided.B6,A/J and BALB/c mice into four groups,respectively,three of which were experiment groups,containing 10 females and 10 males that were 8 to 10 week-old;while the other one was control group,containing 3 females and 3 males that were also 8 to 10 week-old.We gave the three experiment groups intraperitoneal injection with lmL ZY05719 bacterial liquid of 5×107 CFU/mL,1×108 CFU/mL and 5×108 CFU/mL doses,and the control group received the same volume of THB in the same way.Observed and recorded the death condition.For mice that died within 3 days,we dissected their brain tissues and conducted distributed colony counting;for mice that remained alive after 15 days,we determined their antibody titers through blood sampling.Meanwhile,we created pathlogical sections by leveraging mice with typical symptoms,in order to determine the strains of mice that are susceptible and resistance to ZY05719,as well as the proper infectious doses.Our results showed that:under different infectious doses,there existed insignificant difference between the susceptibilities of B6 and BALB/c mice to SS2 infection and both the two strains showed relatively resistance to SS2;when the infectious dose was 1×108 CFU/mL,the difference between the susceptibilities of B6 and A/J mice to SS2 was significant(A/J mice were susceptible and B6 mice were resistant).This work successfully determines the mice strains that are susceptible and resistant to SS2 infection,which sets a solid foundation for screening related genes that are resistant to Swine Anti-Swine Streptococcosis.2.GW AS on Susceptibility of F2 Mice Hybrid from A/J and B6 to Streptococcus suis Type 2Genome-wide association study(GWAS)is an effective method that leverages statistical tools to examine the whole genome-wide genetic association between genetic variants and observable traits.In this work,we selected A/J mice and B6 mice as parents,and hybridized the two stains in non-inbreeding way to obtain F2 mice.For every F2 mouse,we gave it intraperitoneal injection with 1mL ZY05719 of 1×108 CFU/mL.We screened resistant and susceptible samples,totally 90 samples,according to the death condition and other auxiliary indices.We extracted genomic DNA from these samples,and process them after quality control.We obtained significant SNPs from the scanning analysis of Affymetrix(?)Mouse Diversity Genotyping Array.From the Manhattan plots,we observed that there exist significantly-gathered association signals on the qB,qC1.1 cytobands of chromosome 2;compared to other chromosomes,SNPs on qAl,qE3 cytobands of chromosome 7 and qF2 cytobands of chromosome 12 are also significant,which might be related to the susceptibility of the mice to SS2 and needs special attention.3.Gene Expression Profiling Analysis of Different Mice Strains infected Streptococcus suis Type 2Gene chip can be used to perform high-throughput examination of gene functions and expression conditions,as well as analyzing the regulation function of single gene or multiple genes in gene regulatory network.In this work,we selected male A/J and B6 mice that were 8 to 10 week-old,and divided each strain into four groups,3 mice for each.We gave the three groups intraperitoneal injection with lmL 1×108 CFU/mL ZY05719,and sampled their blood after 2,4 and 8 hours of injection;we used the remaining one group as blank control,and sampled blood after injecting the same amount of THB culture fluid.We extracted RNA from blood samples,and processed them after quality control.We obtained differentially expressed genes through GeneChip(?)Mouse Transcriptome Assay 1.0,and performed enrichment analysis and gene ontology analysis.Our results showed that:specific DEGs are involved in signaling by the BCR/TCR,antigen processing and presentation,regulation of immune system process and Staphylococcus aureus infection,which are closely related to immunology and might be relevant to the susceptibility of the mice to SS2.Thus these DEGs should be further studied.4.Analysis of Swine Anti-Swine Streptococcosis Resistance-Related GenesIn order to identify related genes that are resistant to Anti-Swine Streptococcosis,we conducted analysis by leveraging the results of SNP chip and the results of gene express profile chip.We used the 500kb segments above and below significant SNPs as extension interval,and screened differentially expressed genes in that range.We further narrowed down the range of candidate genes that are resistant to Swine Anti-Swine Streptococcosis according to enlarged Manhattan plots and related literature,and verified their functions using CRISPR/Cas9 and targeted genome editing.We found that:(1)ArhGAP15participates in cytoskeleton shape regulation and Racl protein activity inhibition,and is closely related to the bacterial infection;(2)Epnl participates in the clathrin dependent endocytosis,in the form of endocytosis connector;(3)CD55 is able to inhibit the formation of MAC,through classic complement pathway and alternate complement pathway,in order to protect cells from being attacked.We selected these three types of genes as target genes,and used CRISPR/Cas9 technology to knock them out in the cells.Up to now,we have obtained the single clone cell strain which knocked out ArhGAP15,which sets a solid foundation for verifying functions of target genes.