| Gallibacterium was recently established as a new genus within thefamily Pasteurellaceae. Gallibacterium anatis is a pathogen in chickens and otheravian species where it is a significant cause of salpingitis and peritonitis. Diseaseassociated with this microorganism is related to egg peritonitis, dearease in eggproduction, and occasionally increase in mortality. In2003, the establishment of anew genus Pasteurella Division Chicken genus (Gallibacterium), Gallibacteriumanatis (G. anatis) is representative species of the genus chicken. While a host ofillnesses caused due to its similarity with other pathogens,it had not been given auniform naming until2003. Currently, some scholars believed that G. anatis which isa common organism of the upper respiratory and salpingtis of poultry, capable ofrespiratory and lower genital tract and other parts of the body to survive in the host.When the body is subjected to external stimuli, G. anatis proliferate, it may break thebarrier and damage the immune host. A lot of artificial infection with invasive bacteriatests proved that chickens can spread from the site of entry into the visceral organs.However, under the current experimental results, the chicken pathogenic mechanismis still not understood thoroughly. Therefore, we pathogenic bacteria on chicken isstill no unified conclusion. In2010, the Danish scholar Kristensen first discoveredbacteria in vitro chicken ducks secreted proteins (Gallibacterium toxin A, GtxA),which has leukocidin toxin protein activity and hemolytic activity. The research haslaunched G. anatis can infectious in mice for the first time; prokaryotic expression invitro protein GtxA of G. anatis and explore the toxic effects GtxA chicken peripheralblood lymphocytes;expression of G. anatis Toxin GtxA, development of an IndirectELISA for the detection of serum antibody against GtxA in chickens and has beenapplied in the actual production.To understand whether G. anatis pathogenic in mice, the laboratory itself isolated,identified and saved eight strains G. anatis were injected mice, observed changes in disease response and toxicity in mice after the challenge. The results showed thatdifferent strains of the same dilution showed a different virulence, including strainsYU-PDS-RZ-1-SLG. Poison attack death of mouse tissues in various organs obviouslesions sepsis, liver, spleen particularly prominent, followed by the gut, heart, lungand other organ damage is not obvious. The liver and spleen pathological changeswere caused by strain YU-PDS-RZ-1-SLG, strain YU-XX-HJ-4-SLG and strainSHAN-XY-1-GZ, which were more serious. The results showed isolated from theinfected chicken hen oviduct ducks virulent in organs isolated from other strains,indicating that the separation of different parts of the G. anatis on the strength of itsorgans.To research of toxin protein GtxA of G. anatis virulence role, prokaryoticexpression system in E.coli in vitro expression of recombinant proteins GtxA,N-terminal domain and the C-terminal domain of the protein. After in vitro activity ofthe recombinant protein to identify the role of chicken peripheral blood lymphocytes.It was detected extent of the damage cells by MTT assay. The results indicated thatthe expression of recombinant proteins GtxA can not only inhibit the proliferation oflymphocytes, and showed significant cytotoxicity. N-terminal domain as a separateelement not show toxic effects, but can facilitate protein GtxA play toxin toxicity.After being GtxA peripheral blood lymphocytes stimulated protein, making itselfblocked cell growth, inhibit the secretion of cytokines.Currently, G. anatis with multiple serotypes of chicken and poor cross-protectionbetween serotypes. Protein GtxA that may exist between multiple serotypes. Thepartial gtxA gene was amplified by PCR and inserted into the expression vectorpGEX-6P-1.The plasmid pGEX-6P-1-galli was transformed into E.coliBL21(DE3).The infusion protein was expressed when induced by IPTG. Themolecular weight of recombinant protein was about50kDa analyzed by SDS-PAGE,and the immunoreaction activity of the recombinant protein was confirmed byWestern-blotting. An indirect ELISA for detecting antibody against G. anatis wasdeveloped by using expressed GtxA protein as coating antigen. The result showed thatthe optimal coating antigen was0.403μg/well, the optimal dilution of the serum was1:160and the HRP-labeled rabbit anti-chicken IgG was1:2000. Chicken serumsamples from different herds in Henan province were examined with the ELISAdeveloped in present study and the result was compared with that of previouslyreported ELISA by using G. anatis cell lysates as a coating antigen. The total positive rate of samples detected was19.06%(129/680).The result showed that the methodsestablished was specific, sensitive and can be used for clinical detection as well asepidemiology survey of G. anatis. |
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