Porcine Cytotoxicity of Recombinant Pasteurella multocida Toxin and Establishment of Mouse (Mus musculus) Pathological Model
LI Jin-Feng1,2,3, YUAN Jian-Lin1,2,3, ZHAO Qin1,2,3, DU Sen-Yan1,2,3, WU Rui1,2,3, WEN Yi-Pi1,2,3, HUANG Xiao-Bo1,2,3, YAN Qi-Gui1,2,3, CAO San-Jie1,2,3,*
1 ResearchCenter for Swine Disease/College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China; 2 National Teaching and Experiment Center of Animal, Sichuan Agricultural University, Chengdu 611130, China; 3 Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture, Chengdu 611130, China
Abstract：Pasteurella multocida toxin (PMT), as one of the most important virulence factors of Pasteurella multocida, endangers the health of pigs and causes huge economic losses. To identify the toxicity of recombinant Pasteurella multocida toxin (rPMT) against 4 kinds of porcine cells, and construct the pathological model of toxicity against Mus musculus. pCold Ⅰ-toxA vector was constructed and rPMT was soluble expressed. Morphological observation, cell counting kit-8 (CCK-8) detection and Propidium Iodide (PI) staining were used to explore the toxicity of rPMT against PK15 and other 3 kinds of porcine cells. The median lethal dose (LD50) of rPMT to C57BL/6J mice was determined by Karber method, and the histopathological analysis of heart, liver, spleen, lung, kidney and small intestine was carried out. The results showed that rPMT protein (146 kD) was successfully expressed; After treated with rPMT, PK15 cells arose obvious pathological changes, more cells died and cell activity decreased significantly (P<0.05) among the 4 kinds of porcine cell, but the activity of IPEC cells increased significantly (P<0.05); The LD50 of rPMT against C57BL/6J mice was 0.490 ng/g; After the challenge, the intestinal villi of mice fell off; the congestion of kidney and liver was serious; the splenic sinus, which is in the red pulp area of spleen, bled; The number of lymphocytes decreased and a large number of pigments deposited. This study finds that the toxic effect of rPMT against PK15 was the most significant among the 4 kinds of porcine cells; The cytotoxicity model of rPMT against PK15 was successfully constructed and the pathological model of toxicity aginst C57BL/6J mice was successfully constructed, which provides a theoretical basis for further study of the pathogenic mechanism of PMT.
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