Ontogeny Of The Duck Complement System And The Changes Of Riemerella Anatipestifer Infected Duck

Riemerella anatipestifer is the pathogen of infectious serositis of duck and clinical symptoms of the disease showed sepsis with fibrinous pericarditis, perihepatitis, meningitis, airsacculitis and neurological. In addition, it was presented caseous salpingitis, conjunctivitis, arthritis and other symptoms in some cases. It has high morbidity and mortality. Currently, The disease was distributed worldwide.The complement system is crucial to the host defense against invading bacteria, and it is the important component of innate immune system. It’s not only the first line of host defense against pathogens, involved in scavenging "danger signal", but also plays the extremely important role in the regulation of inflammation and immune response in the process.The level of complement activity represents the ability of the complement system to resist invading pathogenic microorganisms. Detection of complement activity and the individual component of complement has an important role on functional evaluation of the immune system, diagnosis and treatment of the disease and epidemiological investigation.In this study, the duck complement activity were detected and the expression of duck C3 d, CR2 was analyzed before and after infected by RA in order to reveal the effect on complement system by the infection of Riemerella anatipestifer. It lays the foundation for further studying the complement in the duck anti-RA immunity. The main results of this study are as follows: 1. The establishment of Sichuan duck RA infection model10th age Sichuan duck under the experimental conditions of feeding were injected subcutaneously RA bacteria 0.2m L / only(1 × 109 cfu / mL) at leg portion,within 168 h after inoculation the morbidity was 80%,mortality rate was 70%, which after inoculation 24 h appeared obvious clinical symptoms, mortality peak appeared in 36h~ 60 h. Dead duck anatomy showed all serous,the capsule of tissues and organs were fibrinous exudated and attached, presented a typical fibrinous pericarditis, liver inflammation; congestion and enlargement of the heart, liver, and spleen. 2. Changes of serum complement activity of Sichuan duckWe used hemolysin sensitized chicken red blood cells and fixation serum dilution(1: 5) to determine the classical pathway complement hemolytic activity of Sichuan duck. The hemolytic activity of the alternative complement pathway was determined by 1% rabbit red blood cells and fixed serum dilution(1:5). The classical pathway complement hemolytic activity of Sichuan duck gradually increased in 1~15d, 15~21d stabilized and close to adult duck; the alternative pathway hemolytic activity gradually increased in 1~15d, 15~21d stabilized and close to adult duck.The classical pathway complement hemolytic activity of 10 th age Sichuan duck infected RA increased in 12~24h after infection, and higher than the control group; gradually reduced in 36~48h, and then gradually increased in 48h~7d, but lower than the control group. The alternative pathway hemolytic activity in serum decreased in 12~48h after infection, gradually increased in 48~168h, but lower than the control group. 3. Changes of Sichuan duck C3 d and CR2 mRNA expressionAccording to the published on Genebank of duck C3 d gene sequence(GenBank accession number: EU847624.1), duck CR2 EST sequence(accession number: DR765735.1),the primers were designed. With liver and spleen extract total RNA as template to establish RT-PCR, cloning and sequencing by Sichuan duck C3 d and CR2 gene fragment of 991 bp and 347 bp respectively. In the NCBI alignment, it is found that the homology of Sichuan duck C3 d gene and Anas platyrhynchos C3 d gene is 99%, Sichuan duck CR2 gene and Pelecanus crispus CR2 gene is 90%.Based on the obtained C3 d and CR2 gene sequences, the quantitative PCR primers were designed. Duck β-actin gene as reference gene, 1 day as a control, were analyzed C3 d and CR2 mRNA expression using 2-△△Ct method.Duck C3 d mRNA expression increased gradually in 1~12d, 12~21d expression stabilizing. Irregular CR2 mRNA expression in between 1.00~2.87 times, 3,6,9,12,15,18,21 d expression are the first 1d 1.73 times, 1.45 times, 2.55 times, 1.68 times, 1.39 times, 1.82 times and 2.87 times.The C3 d mRNA expression of 10 th age Sichuan duck infected RA gradually reduced in 12~48h after infection, and then gradually increased in 48~168h, but lower than the control group;the CR2 mRNA expression gradually reduced in 12~48h after infection, and then gradually increased in 48~168h, but lower than the control group. 4. Changes of protein content of C3 d and sCR2On the basis of duck C3 d ELISA kit and duck soluble leukocyte differentiation antigen 21(CR2/sCD21) ELISA Kit instructions were used to determine the protein content of duck C3 d and sCR2 respectively.The protein content of C3 d in duck serum 1~9d steady, 9~15d increase, 15~21d stabilized. The protein content of CR2 was at a relatively stable level in 1~21d.The C3 d protein content of 10 th age Sichuan duck infected RA was higher than control group in 12~168h after infection;CR2 protein content was higher than control group in 12h~72h after infection,but lower than control group in 168 h after infection.