Effects Of APEC And Its PhoP/Q Deletion Strain On The Expression Of AvBD6 In Chicken Intestine

Avian pathogenic E.coli(Avian pathogenic Escherichia coli,APEC)was proved not only can infect poultry,but also mammals(including humans),which shows that the APEC is the zoonoses potential pathogens.The PhoP/Q is one of the important two-component system of APEC which regulats the key virulence factor.And it plays an important role in APEC-induced defense responses in vivo.Avian ?-defensin have broad antimicrobial spectrum,strong antibacterial ability,mechanism of action and other unique features,which become the field of biomedical research hotspot.Avian ?-defensin 6(AvBD6)is highly expressed in the digestive tract,and has broad antimicrobial activity and thus it plays an important role in innate host defense of chicken.Currently,the relevant interactions of PhoP/Q two-component system of APEC and the ?-defensins has not been reported.So the purpose of this experiment is carried out to investigate the situation of AvBD6 protein distribution and expresson in chicks' intestinal during infected with APEC and its PhoP/Q deletion strain.We used APEC and its PhoP/Q deletion strain to infect the chicks at the age of 14 days,build infection model and observe its pathological changes.The small intestine from the infected chicks were sampled at different time points post infection.Fluorescence real-time quantitative PCR was used to detect the transcription level changes of AvBD6 in the small intestine tissue,and Immunohistochemistry was used to detect distribution of AvBD6 in the small intestine tissue.This study not only lay the foundation for AvBDs in anti-APEC infection,but also provide a theoretical basis for revealing PhoP/Q two-component system induced AvBDs anti-infection regulatory mechanism after chickens infected with APEC.The contents and results are as follows: 1.Established infection model of chicks infection with APEC and its PhoP/Q deletion strain and pathological changes of chicken intestinalThe experiental group was inoculated with 0.5 ml of the APEC and its PhoP/Q deletion strain suspension containing 1×106 cfu/mL through leg,and the control group was given 0.5 ml of PBS.Chicks pathological changes were observed after infection.The results show that there were mucosal swelling,vascular rupture and it even cause hemorrhagic colitis at 48 h after infected with APEC and its PhoP/Q deletion strain.Small intestinal villus were damaged and even fall off,the center axis of the intestinal villi appear more red cells and inflammatory cells,the columnar epithelial cells are lysed.2.AvBD6 gene mRNA transcription level in the chicks of the small intestinal tissue after infected with APEC and its PhoP/Q deletion strainChicks inoculated with APEC and its PhoP/Q deletion strain at the age of 14 days,and than we would collected duodenal,Jejunum,ileum tissue after infection within 48 h(0 h,6 h,12 h,24 h,36 h and 48 h).Using real-time quantitative PCR to detect AvBD6 gene expression patterns during infection.The results showed that after the chicks infected with APEC and its PhoP/Q deletion strain the AvBD6 mRNA relative expression level in the small intestine within 24-48 h was that: the PhoP/Q mutant infection group> the APEC infection group>the blank control group,and it reached its maximum at 48 h.There was a substantially horizontal posture of the AvBD6 mRNA relative expression of the blank control group during 0-48 h.The AvBD6 mRNA relative expression in duodenum and jejunum was higher than that in ileum of blank control group(P<0.05).The AvBD6 relative expression in jejunum of the group which was infected with original strain of APEC is higher than the else within 0-48 h.The AvBD6 mRNA relative expression in duodenum was extraordinary higher than that in jejunum and ileum(P<0.01 or P<0.05).3.The AvBD6 protein expression and location in the chicks of the small intestinal tissue after infected with APEC and its PhoP/Q deletion strainAccording to the results of quantitative PCR,collected duodenum,jejunum and ileum tissue infection at 48 h,using Immunohistochemistry to detect the location of AvBD6 protein in intestine of chicks which was infected with APEC and its PhoP/Q deletion strain at 48 h.The result showed that AvBD6 was mainly distributed in villi epithelial cells and glandular portion of each part of the small intestine.The AvBD6 positive signals in the small intestine tissue brownish yellow.Using Image-pro plus 6.0 software to detect the average optical density value of each tissue section.The result showed that the AvBD6 protein relative expression level in the group which was infected with APEC and its PhoP/Q deletion strain is significantly higher than that in the blank control group at 48 h(P<0.01 or P<0.05).The above experiments show that after chicks infected with APEC and its PhoP/Q deletion strain,the expression and distribution of AvBD6 in chicks in chicks intestine has certain regularity.The AvBD6 mRNA expression level was the highest within 48 h,it showed that: the PhoP/Q mutant infection group> the APEC infection group>the blank control group.In the same condition,The AvBD6 protein in intestine of chicks which was infected with APEC and its PhoP/Q deletion strain was higher than that in the blank control group.This results suggest that AvBD6 plays a role in anti-APEC infection and PhoP/Q may be involved in the regulation of anti-infection effects of AvBD6.