The Pathogenicity Of Cytolethal Distending Toxin Of Haemophilus Parasuis On Cells And Piglets

Haemophilus parasuis is a kind of Gram-negative opportunistic pathogen in swine upper respiratory tract, which is one of the most important pathogens causing death in nursery piglets. The virulence factors and pathogenic mechanisms of H. parasuis is still unclear due to backwardness, which restricts the diagnosis and control of this disease in a certain degree.CDT is the only member of the bacterial AB toxins which exhibits DNase activity. It can cause the DSBs(DNA double-strand breaks) reaction, resulting in irreversible cell cycle arrest and apoptosis in a broad range of eukaryotic cell lineages, and play an important role in the pathogenesis of Gram-negative mucocutaneous bacterial pathogens. Given that the toxin gene cluster is quitely conserved, the cdt gene may be an important virulence factor of H. parasuis. Hence, it is necessary to conduct in-depth research on its pathogenic role.Givens this, focusing on the cdt gene as the object in this research, to explore the pathogenic role of CDT through the cell model in vitro and the piglets model in vivo. The main research results are as follows:1. Construction of cdt mutants by natural transformation in H. parasuisFirst, clone the upstream and downstream homology arms of cdt and gm or kan resistance cassette. Then, connect the homology arms and corresponding resistance cassette by overlapping PCR, and connect them with the vector of p K18 mobsac B to obtain the recombinant plasmid p K18-cdt1-UGD and p K18-cdt2-UKD. Subsequently, the JS0135?cdt1::gm?cdt2::kan strain was generated by natural transformation twice. The cdt mutant named JS0135?cdt1?cdt2 was further identified through PCR, Southern blot and DNA sequencing.2. CDT has a toxic effect on PK-15 cellsThe PK-15 cells were incubated with supernatant protein of JS0135 and the mutants with different doses for 3h. The cells was subsequently cultured for 24 h, 48 h, 7cell morphology was observed on PK-15 with secreted proteins of JS0135: cell sparexpansion of the cell size and nucleus, hyperchromatic nuclei, cell vacuoles, aeven disintegration, compared with the cdt mutants and the blank control. And this toeffect is time- and dose-dependent. These observation indicated qualitatively the toeffect of CDT on PK-15 cells.At the same time, the PK-15 cells were incubated with JS0135 and the cdt mutant for 4h and 6h. After that the lactate dehydrogenase(LDH) activity was detected in the culture supernatant using LDH cytotoxicity kit. We found the cytotoxicity of wild strain isstronger than cdt mutant strain, and the difference was extremely significant. These observations indicated quantitatively the toxic effect of CDT on PK-15 cells.3. CDT is involved in the resist of phagocytosisThe porcine alveolar macrophages cell 3D4/2 were incubated with JS0135 and thecdt mutant strain. The results showed that the resistant phagocytic capacity of JS0135 wasstronger than the cdt mutant strain, suggesting that CDT is related to resist phagocytosisin H. parasuis.4. CDT plays certain role in the pathogenesis in pigletsThe piglets were pleural challenged with JS0135 and the cdt mutant strain in three different doses. Then the virulence differences between wild and mutant strain was compared by the index of clinical symptoms, autopsy, histological, and pathological sections. The results showed that the virulence of the cdt deletion mutant was decreased slightly compared with the wild strain, not as significantly as expected.