|
|
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.
|
Received: 15 February 2022
|
|
Corresponding Authors:
*csanjie@sicau.edu.cn
|
|
|
|
[1] 王豪男. 2017. 我国部分省区猪源多杀性巴氏杆菌的分子流行病学调查[D]. 硕士学位论文, 华中农业大学, 导师:吴斌, pp. 1-70. (Wang H N.2017. Molecular epidemiology of Pasteurella multocida from swine in some provinces of China[D]. Thesis for M.S., Huazhong Agricultural University, Suppervisor: Wu B, pp. 1-70.) [2] 吴中华. 2016. Triton X-114用于去除内毒素的研究[J]. 安徽医药, 20(5): 848-851. (Wu Z H.2016. Removal of endotoxin by triton x-114 phase separation[J]. Anhui Medical and Pharmaceutical Journal, 20(5): 848-851.) [3] 熊款款, 吴映欣, 易思亮, 等. 2021. T-2毒素致猪肾细胞损伤及氧化应激相关机理的研究[J]. 中国畜牧兽医, 48(12): 4710-4717. (Xiong K K, WU Y X, Yi S L, et al.2021. Mechanism of T-2 toxin induced injury and oxidative stress in PK15 cells[J]. China Animal Husbandry & Veterinary Medicine, 48(12): 4710-4717.) [4] 尹媛媛, 何芳, 赵光夫, 等. 2021. 多杀性巴氏杆菌主要毒力因子研究进展[J]. 中国兽医学报, 41(06): 1210-1218. (Yin Y Y, He F, Zhao G F, et al.2021. Research progress on the main virulence factors of Pasteurella multocida[J]. Chinese Journal of Veterinary Science, 41(06): 1210-1218. [5] 周瑶. 2021. 猪多杀性巴氏杆菌病的诊断与防控措施[J]. 现代畜牧科技, (12): 112-113. (Zhou Y. 2021. Diagnosis and control measures of Pasteurellosis multocida in pigs[J]. Modern Animal Husbandry Science & Technology, (12): 112-113.) [6] Arumugam N D, Ajam N, Blackall P J, et al.2011. Capsular serotyping of Pasteurella multocida from various animal hosts-a comparison of phenotypic and genotypic methods[J]. Tropical Biomedicine, 28(1): 55-63. [7] Cheville N F, Rimler R B.1989. A protein toxin from Pasteurella multocida type D causes acute and chronic hepatic toxicity in rats[J]. Veterinary Pathology, 26(2): 148-157. [8] Copeland S, Warren H S, Lowry S F, et al.2005. Acute inflammatory response to endotoxin in mice and humans[J]. Clinical & Diagnostic Laboratory Immunology, 12(1): 60-67. [9] Elling F, Pedersen K B.1985. The pathogenesis of persistent turbinate atrophy induced by toxigenic Pasteurella multocida in pigs[J]. Veterinary Pathology, 22(5): 469-474. [10] Gan F, Zhou Y, Hou L, et al.2017. Ochratoxin A induces nephrotoxicity and immunotoxicity through different MAPK signaling pathways in PK15 cells and porcine primary splenocytes[J]. Chemosphere, 182(sep.): 630-637. [11] Hildebrand D, Heeg K, Kubatzky K F.2011. Pasteurella multocida toxin-stimulated osteoclast differentiation is B cell dependent[J]. Infection and Immunity, 79(1): 220-228. [12] Horiguchi Y.2012. Swine atrophic rhinitis caused by Pasteurella multocida toxin and Bordetella dermonecrotic toxin[J]. Current Topics in Microbiology and Immunology, 361: 113-129. [13] Kamp E M, Van D, Tetenburg B J.1987. Purification of a heat labile dermonecrotic toxin from culture fluid of Pasteurella multocida[J]. Veterinary Microbiology, 13(3): 235-248. [14] Li S, Ran X Q, Xu L, et al.2011. microRNA and mRNA expression profiling analysis of dichlorvos cytotoxicity in porcine kidney epithelial PK15 cells[J]. DNA & Cell Biology, 30(12): 1073-1083. [15] Liao C M, Huang C, Hsuan S L, et al.2006. Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs[J]. Vaccine, 24(1): 27-35. [16] Marina H, Boyce J D, Ben A.2006. Pasteurella multocida pathogenesis: 125 years after Pasteur[J]. FEMS Microbiology Letters, 265(1): 1-10. [17] Nakai T, Sawata A, Tsuji M, et al.1984. Purification of dermonecrotic toxin from a sonic extract of Pasteurella multocida SP-72 serotype D[J]. Infection & Immunity, 46(2): 429-434. [18] Okay S.2015. Comparative genome analysis of five Pasteurella multocida strains to decipher the diversification in pathogenicity and host specialization[J]. Gene, (08): 58-72. [19] Orth J H, Aktories K, Kubatzky K F.2007. Modulation of host cell gene expression through activation of STAT transcription factors by Pasteurella multocida toxin[J]. Journal of Biological Chemistry, 282(5): 3050-3057. [20] Orth J H, Lang S, Taniguchi M, et al.2005. Pasteurella multocida toxin-induced activation of RhoA is mediated via two families of Gα proteins, Gαq and Gα12/13[J]. Journal of Biological Chemistry, 280(44): 36701-36707. [21] Pennings A, Storm P K.1984. A test in vero cell monolayers for toxin production by strains of Pasteurella multocida isolated from pigs suspected of having atrophic rhinitis[J]. Veterinary Microbiology, 9(5): 503-508. [22] Redfield R J, Findlay W A, Bossé J, et al.2006. Evolution of competence and DNA uptake specificity in the Pasteurellaceae[J]. BMC Evolutionary Biology, 6(1): 1-15. [23] Wilson B A, Ho M.2010. Recent insights into Pasteurella multocida toxin and other G-protein-modulating bacterial toxins[J]. Future Microbiology, 5(8): 1185-1201. [24] Wilson B A, Ponferrada V G, Vallance J E, et al.1999. Localization of the intracellular activity domain of Pasteurella multocida toxin to the N terminus[J]. Infection & Immunity, 67(1): 80-87. [25] Wu M C, Lo Y T, Wu H C, et al.2021. Cross-protection of recombinant Pasteurella multocida toxin proteins against atrophic rhinitis in mice[J]. Research in Veterinary Science, 137: 138-143. |
|
|
|