Preliminary Study On The Ability Of Biofilm Formation And Effects On Chicken Oviduct Epithelial Cells Of Gallibacterium Anatis

Gallibacterium is a relatively new genus within the family Pasteurellaceae, and Gallibacterium anatis(G.anatis) is the type species. G. anatis constituted a part of the normal flora of the upper respiratory tract as well as the lower genital tract of chickens, but also has potential to cause salpingitis and peritonitis in egg-layer, leading to decreased egg production and increased mortality worldwide. There were a few studies on pathogenic characteristic of G. anatis in experimentally infected chicks in recent years. However, it is still unknown about the factors of affecting biofilm formation and effects on chicken oviduct epithelial cells of G. anatis. In this study, we aim to investigate the factors of affecting biofilm formation and establish an efficient method for isolation and culture of the normal chicken oviduct epithelial cells(COECs) in vitro for studying the pathogenicity and invasion characteristic of G. anatis.To investigate the biofilm formation of G. anatis, this study observed 20 strains of biofilm formation by establishing quantitative adhesion test in vitro and a strong biofilm formation strain was studied under different culture times, mediums, substratums and nutrients. The results showed that the significant biofilms were formed completely by 2 out of 20 strains, while 3 strains were moderate biofilm formation. Improved plate method, tube test and 96-well microtiter plate assays showed that biofilm was well developed after 24 h, 60 h and 24 h cultivation, respectively. The formation period included an initial adhesion to the surface at 8h, the conglutination of micro-colonies at 20 h, the mature of biofilm at 24 h, and the desquamation from the substratum at 32 h, followed by a new formation circulation. Glucose, sucrose and Na Cl inhibited the biofilm formation, while low concentration of Mg SO4 would enhance the development of biofilm formation.Scanning electron microscopy revealed that G. anatis gathered into groups, and a dense three-dimensional structure were formed.To establish an efficient method for isolation and culture of the normal COECs in vitro for studying the pathogenicity and invasion characteristic of G. anatis, different factors were tested to optimize COECs primary culture for repeatable results: the origin of isolated cells(oviduct Infundibulum or Magnum section), the digestive enzymes, tissue digestion times, and the culture plates coating. Identification was performed by transmission electron microscope. The COECs isolated by mincing the infundibular neck and digestion of tissue for 15 min with 0.25% trypsin + 0.02% EDTA, then removing single cells by low-speed centrifuge and fibroblast cells by differential velocity adherent formed cell aggregates of bright colour which was the best result obtained from all applied procedures in our studies. The COECs attached within 24 h, proliferated significantly within 48~60h, gave polygonal pavestone-like monolayer of epithelial-like character and developed slowly after 72 h. Such colonies can survive for more than 10 d, and cells grew well after passaging. The cultured cells were identified as COECs by Giemsa staining and Transmission electron microscope. This established method can obtained high purity COECs in vitro.To evaluate the properties of G. anatis on primary COECs, adhesion and invasion experiment, Giemsa staining and scanning electron microscopy(SEM) were applied to determine the difference between strains Yu-PDS-RZ-1-SLG and F149 T. The results showed that G. anatis strain Yu-PDS-RZ-1-SLG adhered to COEC at a higher level compared to G. anatis F149 T and no invasion was observed in Gentamicin resistance assay, Giemsa staining and SEM. However, cell debris and cell apoptosis were observed after being exposed to G. anatis Yu-PDS-RZ-1-SLG for 90 min, while G. anatis F149 T caused no cell damage. Adherence was prevented by treating bacterial cells with trypsin, suggesting the participation of proteins in this process. The levels of cytokines was detected by ELISA after infection, and the results showed that IL-6, TNF-α and IFN-γ levels of treatment group were higher than control group. Changes of cytokines suggested that the production of cytokines might influence the microenvironment of oviduct, and promote adherence and invasion, served as a possible mechanism causing cell damage.To explore the mechanism of toxin protein Gtx A of G. anatis on primary COECs, in vitro expression of recombinant proteins Gtx A, N-terminal domain and the C-terminal domain of the protein were infected with COECs. It was detected the damage cells by cck-8 and cytokines assay. Results indicated that the recombinant proteins Gtx A can not only inhibit the proliferation of COECs, and showed obvious cytotoxicity. N-terminal domain as a separate element not show toxic effects, but can facilitate protein Gtx A play toxin toxicity. After being infected with Gtx A, COECs secreted more inflammatory cytokines of IL-6, TNF-α and IFN-γ. These suggested that G. anatis adhered to the cell surface, secreted exotoxin causing cell permeability changes, induced the expression of inflammation regulatory factor, and eventually cause cellular damage.