Establishment Of Real Time Quantitative PCR Method For Pasteurella Multocida And The Role Of NLRC3 In Regulating Its Proliferation In Rabbits

Rabbit pasteurellois is an acute septic infectious disease caused by Pasteurella multocida(P.multocida),which is one of the most important infectious diseases in rabbits.It is mainly characterized by acute hemorrhagic sepsis and it can also cause rhinitis,pneumonia,conjunctivitis and so on.Rabbit pasteurellois was reported in most countries and regions,causing economic losses to the rabbit farming.Due to restriction on the use of antibiotics,the threat of bacterial diseases to the aquaculture industry is getting more serious.In this study,a SYBR Green ? Real-Time quantificative PCR(RT-qPCR)method to detect P.multocida was established at the first.This method was applied to the study of the pathogenicity of P.multocida to rabbits and confirmed its colonization in vivo.The host innate immune system is the first line of defense against invasion of pathogenic microorganisms.Nucleotidebinding oligomerization domain-like receptors(NLRs)are an important family of pattern recognition receptors(PRRS),which are key factors of mediating inflammatory response to remove pathogenic microorganisms.For this reason,this study focused on the inflammatory response activated by P.multocida through experimentation in vivo and vitro.This study explores the regulatory mechanism of rabbit NLRC3(rNLRC3)receptor in the P multocida infection and its effect on bacterial proliferation,which provides a research basis for further research on whether it can be used as a new drug or action target.1.Establishment of real time quantitative PCR method for P.multocida and study on pathogenicity of P.multocida to rabbitsThe specific primers were designed based on the sequence of P.multocida 16S rRNA gene,and the standard curve and the melting curve were established.The linear equation of the standard curve is Y=-3.119X+31.4,the amplification efficiency was 1.127 and the correlation coefficient of the reaction is 0.9889,The melting curve proves that a single melting peak;The results of specificity test showed that this method could specifically detect P.multocida;The results of sensitivity test showed that the detetion limit was 1.78× 101CFU/mL,which was 100 times more sensitive than the conventional PCR;The results of repeatability test showed that the coefficient of variation was less than 2%in intra and inter group.In order to explore the pathogenicity of P.multocida to rabbits,the challenge experiment was carried out by using subcutaneous injection of P.multocida dose of 106 CFU of each rabbit.The clinical symptoms and pathological changes of rabbits were observed.The established qRT-PCR method was used to detect the content and distribution of bacteria in various tissues and organs after challenge.The results showed that respiratory symptoms dyspnea and serous nasal fluid were found in poison contrast group.Autopsy showed pulmonary abscess and pulmonary consolidation;the tracheal ring had hemorrhage;the thymus had obvious bleeding points.The section of pathologic tissues was shown pulmonary edema and neutrophil infiltration appeared in the lungs,the epithelial cells of tracheal mucosa were necrotic and exfoliated,and the tracheal ring was congested,the number of lymphocytes decreased and cell necrosis appeared in thymus.The qRT-PCR results showed that the bacterial content reached the peak in most tissues on the second day after challenge and in order from more to less was spleen,liver,thymus,heart,lung,kidney.The bacterial content began to decrease on the third day after challenge.The expression change of the inflammatory factor and pattern recognition receptors in thymus,lung and kidney after challenge was detected by qRT-PCR.The results showed that the expression of IL-1?,IL-6,IL-8 and TNF-? was significantly up-regulated(P<0.05)in all tested tissues,and was the highest on the second day after challenge,and then began to be down regulated.The expression of TLR2 and TLR4 was significantly(P<0.05)up-regulated after infection,which indicated that P.multocida activated inflammatory response by TLR2 and TLR4.The expression level of rNLRC3 was inhibited at the early stage of infection and increased at third days after infection,which was negatively correlated with the expression of inflammatory factors.We detected the expression of rNLRC3 in rabbit kidney cellscells after P.multocida stimulation at the same time.The results showed that the expression of rNLRC3 decreased within 10 hours of infection,and then gradually recovered,which was consistent with the results of in vivo experiments.Therefore,we speculated that rNLRC3 may have the function of negatively regulation of inflammatory response induced by P.multocida.2.The role of rNLRC3 in regulating the proliferation of P.multocidaIn order to further explore the role of rNLRC3 during P.multocida infection,this study is the first time.to clone rNLRC3 gene and constructed eukaryotic expression vector.rNLRC3 protein was located in the intracytoplasm in RK-13 and do not colocalize with the mitochondria by laser scanning confocal microscopy observation.Western blot was used to detect the effect of overexpress and knockdown of rNLRC3 on P65 phosphorylation and nuclear import induced by P.multocida.The results showed that the level of P65 phosphorylation decreased significantly(P<0.05)and nuclear translocation reduced after overexpress rNLRC3,while the level of P65 phosphorylation increased after knockdown of rNLRC3.The expression of IL-1??IL-6 induced by P.multocida significantly decreased after overexpress rNLRC3,while the expression of IL-8 were significantly increased(P<0.05)after knockdown of rNLRC3.The effect of rNLRC3 on the proliferation of P.multocida was detected by qRT-PCR.The results showed that the bacterial proliferation increased significantly(P<0.05)after overexpress rNLRC3,while the bacterial proliferation decreased significantly(P<0.05)after knockdown rNLRC3.It indicated that rNLRC3 can promote the proliferation of P.multocida.through negatively regulation of NF-?B pathway,and knockdown rNLRC3 inhibited the proliferation of bacteria through promoting activation of NF-?B.Finally,the functional domain of rNLRC3 was confirmed.The results showed that the NACHT-LRR of rNLRC3 inhibited P65 phosphorylation and reduced the expression of IL-1?,IL-6,IL-8 and TNF-?.It indicated that the functional domain of rNLRC3 is NACHT-LRR.