Phyletic Evolution Analysis, Distribution Regularity In Chick And The Subunit Vaccine Development Of Proteus Mirabilis

Proteus mirabilis widely distributed in nature within the Proteus of Enterobacteriaceae,commonly found in sewage, soil and compost, is a common saprophytic opportunisticpathogen. In humans, the bacteria has been one of the main cause of food poisoning andshowed an increasing trend in recent years. In1996, it was reported that the bacteria couldcause diease in chickens and since then the related report constantly appeared. Proteusmirabilis can cause many clinic symptoms in chickens, including poultry diarrhea, paralysisof the limbs, sepsis and encephalomalacia. The disease is infected in various day-old chickens,especially within4-week-old chicks. The disease spreads by horizontal transmission orvertical transmission, and can easily lead to outbreak of epidemic diseases. Therefore, in orderto explore the pathogenesis and prevention methods of Proteus mirabilis, the thesis explainsthe study from three aspects.1. Identification and Phyletic evolution analysis of Proteus mirabilis strains by PCRand restriction fragment length polymorphism of16S-23S rRNA gene intergenicspacer regionTo establish a new method to identify Proteus mirabilis strains, according to theconserved sequences of16S and23S which located on both ends of the clinical commonpathogenic bacteria16S-23S rRNA gene intergenic spacer region (ISR), a pair of universalprimers was designed. Nine Proteus mirabilis strains and six similar bacteria strains wereamplified by PCR and identified by the PCR length polymorphism comparison, restrictionfragment length polymorphism (RFLP) analysis and partial sequences sequencing. The resultshowed that Proteus mirabilis strains could be discriminated from other similar bacteriastrains by PCR length polymorphism comparison, all of test organism could be discriminatedby RFLP and Proteus mirabilis strains could be typed by partial sequences sequencing. Theresult indicated that the identification method based on the16S-23S rRNA ISR, using PCRand PCR-RFLP, is very suitable for the rapid low-cost identification and discrimination ofProteus mirabilis strains from other phylogenetically related bacteria strains.2. Distribution and dynamic diversity of Proteus mirabilis in chickensProteus mirabilis strain Q1challenged the3-day-old chicks to establish the animal models of the Proteus mirabilis disease. At8h,16h,1d,2d,3d and7d after challenge, theheart, liver, spleen, lung, kidney, bursa, gizzard, duodenum and cerebellum were collected andquickly placed in4%poly-formaldehyde to fix. Enzyme immunohistochemistry method wasestablished to detect the fixed tissues and organs. The results show that the colonization orderof Proteus mirabilis in tissues and organs is: the duodenum, cerebellum, bursa, spleen>liver,lung>kidney; At1-3d, the bacteria most widely spreaded, but it most spreaded in theduodenum and cerebellum; At7d, a small amount of bacteria were detected in the duodenumand cerebellum of the surviving animals; The bacteria colonized in the parenchymal cells andepithelial cells; The bacteria was detected in all plates coated. The results indicate that theduodenum and cerebellum are the majou garget organs for the bacteria, parenchymal cells andepithelial cells are the major target cells for the bacteria, and the bacteria can enter thecirculatory system to cause bacteremia or sepsis. These results explain the distribution ofProteus mirabilis in chickens and the pathogenesis of the disease, which laid a foundation forthe study of the prevention and treatment of the disease.3. Effects of Taishan Pinus massoniana pollen polysaccharide on the subunit vaccine ofProteus mirabilis in chickensThree adjuvants, namely, Taishan Pinus massoniana pollen polysaccharide (TPPPS),white mineral oil (WO) and propolis (PP), were added to the outer membrane protein (OMP)of Proteus mirabilis (P. mirabilis) and their effects were compared. Three hundred1-day-oldchicks were randomly divided into five groups (I–V), with60chicks per group, and injectedsubcutaneously with WO-OMP vaccine (I), PP-OMP vaccine (II), TPPPS-OMP vaccine (III),OMP-only vaccine (IV) and physiological saline (V) at3,7and12days old. On days3,7,14,21,28,35,42and49after the first vaccination, the antibody titers, interleukin-2levels (IL-2)and T-lymphocyte proliferation rates in the peripheral blood as well as the secreting-type IgAlevels (SIgA) in the duodenum were measured. On day7after the third vaccination, thechicks were challenged with P. mirabilis strain Q1and the protective effects of each groupwere observed. The highest protective rate was observed in group III. Moreover, the antibodytiters as well as IL-2, SIgA and T-lymphocyte proliferation rates in this group significantlyincreased and were significantly higher than those in the other groups at most time points. Theresults indicate that TPPPS could significantly enhance the effects of the subunit vaccine of P.mirabilis; induced stronger humoral, cellular and mucosal immunity as compared with WOand PP; and should be developed as a vaccine adjuvant.