NF-κB signaling pathway mechanism in cow intertoe skin inflammation caused by Fusobacterium necrophorum.

Background: Fusobacterium necrophorum is the main pathogen inducing bovine foot rot. The infected site is often accompanied by a strong inflammatory response, but the specific inflammatory regulatory mechanism remains unclear. Aim: A cow skin explants model was established to elucidate the mechanism of F. necrophorum bacillus causing foot rot in cows, and to provide reference for future clinical practice. Methods: Cow intertoe skin explants were cultured in vitro, and F. necrophorum bacteria solution and nuclear factor-κB (NF-κB) inhibitor BAY 1-7082 were added to establish an in vitro infection model. Hematoxylin and eosin staining, terminal - deoxynucleotidyl transferase mediated nick end labeling, and immunohistochemistry were used to detect the pathological changes of the skin explants infected with F. necrophorum, the degree of tissue cell apoptosis, and the expression of the apoptosis-related protein Caspase-3, respectively. RT-qPCR, Western blot, and ELISA were used to detect the activation of the NF-κB pathway and inflammatory cytokines by F. necrophorum. Results: The intertoe skin structure of cows infected with F. necrophorum changed with different degrees of inflammation, and the degree of tissue cell apoptosis was significantly increased (P < 0.01). In addition, infection with F. necrophorum significantly increased the phosphorylation level of IκBα protein and up-regulated the expression level of NF-κB p65. The high expression and transcriptional activity of NF-κB p65 significantly increased the expression and concentration of the inflammatory cytokines TNF-α, IL-1ß, and IL-8, thus inducing the occurrence of an inflammatory response. However, inhibition of NF-κB p65 activity significantly decreased the expression of inflammatory factors in the intertoe skin of cows infected with F. necrophorum. Conclusion: F. necrophorum activates NF-κB signaling pathway by increasing the expression of TNF-α, IL-1ß, IL-8 and other inflammatory factors, leading to foot rot in dairy cows.

The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate).

Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.

Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Partial characterization of leukocidin from Fusobacterium necrophorum.

Leukocidin from Fusobacterium necrophorum was produced in the diffusate of a dialysis culture. It was free from deoxyribonuclease, fibrinolysin, gelatinase, haemolysin, lipase, caseinase and endotoxin. The leukocidin had a molecular weight between 10,000 and 5,000 as estimated by membrane partition chromatography. It formed precipitin lines with anti-leukocidin-serum in double immunodiffusion tests. Mouse peritoneal cells were characteristically damaged by the leukocidin, as revealed by scanning electron microscopy. The damaged cells lost microvilli and suffered partial destruction of their cell membranes.

Comparative in vitro leukotoxin production of three bovine strains of Fusobacterium necrophorum.

The in vitro leukotoxic activity of 3 bovine isolates of Fusobacterium necrophorum which varied in pathogenicity were compared. Monolayers of mouse peritoneal macrophages were exposed to culture filtrates from each F necrophorum strain, and cell viability was determined, using the trypan blue dye exclusion test. Two methods were used for production of the leukotoxin: (1) medium M-1 continuous dialysis sac cultures and (2) brain-heart infusion agar plate cultures. Supernatant cultural fluids containing the leukotoxin were subjected to membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. All ultrafiltrates had a cytotoxic effect on the monolayers. Cytotoxic activity was not found in the ultrafilter residues or in the control media ultrafiltrates. Comparative study of leukotoxin production indicated that F necrophorum 2101, type A, produced the most leukotoxin; F necrophorum 2030, type AB, produced slightly less leukotoxin; and F necrophorum 2035, type B, produced small amounts of leukotoxin. Endotoxin activity, as demonstrated by the mouse lethality test, was found in the residues of the XM-100A ultrafilter (100,000 mol wt), but not in the filtrates. Culture supernatant fluids and the XM-100A ultrafiltrates were positive for endotoxin, using the limulus amebocyte lysate assay; however, the other ultrafiltrates with lower mol wt exclusion limits were negative.