title: Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines.
abstract: In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in whole-blood culture at 4h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-α and IL-1β changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-α in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test.
authors: National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1, Tokura, Kokubunji, Tokyo 185-8511, Japan. saiki_imamura@nval.maff.go.jp
authors: Saiki
authors: S
authors: Imamura
authors: Mari
authors: M
authors: Nakamizo
authors: Michiko
authors: M
authors: Kawanishi
authors: Nao
authors: N
authors: Nakajima
authors: Kinya
authors: K
authors: Yamamoto
authors: Mariko
authors: M
authors: Uchiyama
authors: Fumiya
authors: F
authors: Hirano
authors: Hidetaka
authors: H
authors: Nagai
authors: Mayumi
authors: M
authors: Kijima
authors: Ryoyo
authors: R
authors: Ikebuchi
authors: Hirohisa
authors: H
authors: Mekata
authors: Shiro
authors: S
authors: Murata
authors: Satoru
authors: S
authors: Konnai
authors: Kazuhiko
authors: K
authors: Ohashi
mesh_terms: D000536:Aluminum Hydroxide / Q000494:pharmacology; D000818:Animals; D001428:Bacterial Vaccines / Q000276:immunology*; D002417:Cattle; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000276:immunology*; D016753:Interleukin-10 / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D001428:Bacterial Vaccines; D016207:Cytokines; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D000536:Aluminum Hydroxide