Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice

Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.     

Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leucocidin

The capacity of extracts from toxigenic and non-toxigenic ruminant strains of Fusobacterium necrophorum to protect against challenge with homologous and heterologous bacteria was examined in mice. The numbers of F. necrophorum which were infective or lethal for mice increased 5- to 8-fold in animals which had been previously inoculated with complete Freund's adjuvant (FCA). Although preparations containing lipopolysaccharide (LPS) and outer membrane proteins (OMP) from several strains gave protection against a non-toxigenic strain (FnB-3), they did not significantly immunize mice against a challenge infection with a toxigenic bovine strain, FnB-1. Only material which had been prepared by gel filtration of 18-h liquid culture supernates of toxigenic F. necrophorum elicited significant immunity against homologous challenge with FnB-1. This preparation contained LPS and the majority of the leucotoxic activity. However, passive protection was not afforded to mice inoculated with bovine or rabbit sera which possessed high neutralization titres against the leucocidin.     

Serological response of mice to Fusobacterium necrophorum

Mice immunised with killed or living Fusobacterium necrophorum, by five different regimens, almost invariably failed to produce antibodies demonstrable by a passive haemagglutination test. An enzyme-linked immunosorbent assay (ELISA), however, usually demonstrated a serum antibody response. This suggested that F necrophorum was not in fact immunosuppressive in mice--a possibility that had been entertained to explain the great difficulty in protecting mice immunologically against challenge with F necrophorum.     

Comparative biological features of a rat liver abscess model induced with three Fusobacterium necrophorum strains

Several biological features were compared in a rat liver abscess model, using intraportal inoculations with 3 bovine strains of Fusobacterium necrophorum which varied in virulence. Serum alanine aminotransferase activities were increased significantly (P less than 0.05) in rats inoculated with F necrophorum 2101 by postinoculation hours 6, 12, and 24. Thereafter, alanine aminotransferase values returned to base line for the remainder of the experiment. Also, rats inoculated with F necrophorum 2101 had a significantly greater (P less than 0.05) weight loss than did the control rats during the first 5 postinoculation days and developed leukocytosis characterized by a neutrophilia with a left shift. The duration of the bacteremia was related directly to the virulence of the F necrophorum strain. Fusobacterium necrophorum 2101, a biotype A which was the most virulent, induced the most persistent bacteremia; F necrophorum 2035, a biotype B which was the least virulent, produced the shortest bacteremia; and F necrophorum 2030, a biotype AB which was of intermediate virulence, led to bacteremia of intermediate duration. Plasma endotoxin was demonstrated intermittently during the first 24 hours, but did not correlate with the bacteremia.     

Immunohistochemical and ultrastructural identification of Fusobacterium necrophorum subsp. necrophorum in bovine fatal necrotizing glossitis

A 37-day-old male Japanese black calf showing marked salivation and leucocytosis died and was examined the tissues histologically. Histological lesions were characterized by severe focal necrotic glossitis on the ventral side of the root of the tongue. Immunohistochemically, Fusobacterium necrophorum subsp. necrophorum antigen was detected in the necrotic tissues and its distribution corresponded to that of the gram-negative, nonsporeforming, long filamentous organisms. Ultrastructural similarities between the organism and F. necrophorum subsp. necrophorum, but not subsp. funduliforme were observed. These findings clearly demonstrated that the fatal necrotic glossitis was caused by F. necrophorum subsp. necrophorum. This is the first report of bovine fatal necrotizing glossitis with leucocytosis caused by F. necrophorum subsp. necrophorum infection, and this organism may be an important fatal pathogen in calves with glossal lesions.     

Endotoxin from Fusobacterium necrophorum of bovine hepatic abscess origin.

The endotoxic activity of Fusobacterium necrophorum bov 5 was investigated. The supernatant (S) fluid and cell wall (CW) preparation, obtained after differential centrifugation of the ruptured cell mass, were lethal for mice. The toxicity of the S fluid was stable during prolonged storage, treatment with formalin, and heating for 15 minutes at 80, 100, and 121 C, but was destroyed by alkaline hydrolysis with 0.25 N NaOH. The toxic factor was found in a high molecular weight (MW) fraction after gel filtration. The properties exhibited by the toxic S fluid resembled those of endotoxic lipopolysaccharide (LPS). Extracted and partially purified LPS (endotoxin) from F necrophorum bov 5 demonstrated a mouse median lethal dose (mouse LD50) of 16.8 mg/kg of body weight. The toxic LPS material, a high molecular weight moiety as estimated by gel filtration, was resistant to ribonuclease (RNase), deoxyribonuclease (DNase), and pronase treatment. A positive Shwartzman reaction (median skin lesion dose (SLD50) equal to 3.32 mug/kg of body weight) and biphasic fever response (minimal dose required to produce a fever index of 40 sq cm which falls on the linear portion of dose-response curve (FL40) equal to 0.41 mug/kg of body weight) further indicated the toxin was endotoxin in nature. The LPS from F necrophorum bov 5 was less toxic than Salmonella typhimurium LPS; but had considerable toxicity for experimental animals. The toxic activity of the partially purified F necrophorum bov 5 endotoxin was separated into 2 fraction regions by diethylaminoethyl (DEAE)-cellulose chromatography. The data provide evidence for the production of a potent endotoxin, possibly composed of more than one toxic component, which may be released upon cell disruption.     

Enzyme-linked immunosorbent assay for detection of milk immunoglobulins to leukocidin toxin of Staphylococcus aureus

Leukocidin toxin from a bovine strain of Staphylococcus aureus was partially purified by ion exchange chromatography. An enzyme-linked immunosorbent assay was developed to quantitate antibodies specific for leukocidin in bovine milk. This was used to assay quarter samples from 88 cows in a S aureus-infected herd for antibody levels to the toxin. Milk samples from 65 cows with S aureus infections in at least one quarter produced a mean optical density of 1.054, whereas milk samples from 23 cows that were free of bacteria on cultural examination had a mean optical density of 0.584. There was a significant difference (P less than 0.001) in milk anti-leukocidin levels between these 2 groups. Evaluation of serum samples from 40 of these cows indicated that the milk anti-leukocidin concentrations were reflective of systemic anti-leukocidin values. The capability of 57 milk samples to neutralize the cytolytic effect of minimal amounts of leukocidin on bovine peripheral blood neutrophils was examined. Good correlation existed between the enzyme-linked immunosorbent assay antibody concentration and toxin-neutralizing capability of individual milk samples.     

Fusobacterium necrophorum hemolysin in bovine hepatic abscess

An immunofluorescence study was made on bovine hepatic abscess containing Fusobacterium necrophorum predominantly. The abscess section stained with anti F. necrophorum hemolysin serum demonstrated fluorescence which formed irregular and granular shapes. Actinomyces pyogenes isolates from the abscess were not stained with the serum. These findings suggest that the bacterial hemolysin contributes to the formation of the hepatic abscess during an infection with F. necrophorum.     

Susceptibility of wallabies to Fusobacterium necrophorum

Wallabies (Macropus rufogriseus) were not appreciably more susceptible than rabbits or mice to Fusobacterium necrophorum, a fact established by the subcutaneous injection of a series of graded doses into animals of each species. The strikingly frequent occurrence of necrobacillosis in captive macropods is therefore not due to a uniquely high susceptibility. A vaccine containing inactivated F necrophorum culture emulsified with Freund's complete adjuvant failed to increase the resistance of wallabies to subcutaneous challenge with a moderate dose of the homologous strain. The control of necrobacillosis in captive wallabies must therefore depend on managemental measures aimed at minimising faecal contamination of the environment and damage to the buccal mucous membrane and skin.     

Factors affecting the leukotoxin activity of Fusobacterium necrophorum.

The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.     

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.     

Failure to induce in rabbits effective immunity to a mixed infection of Fusobacterium necrophorum and Corynebacterium pyogenes with a combined bacterin.

Failure to induce in rabbits effective immunity to a mixed infection of Fusobacterium necrophorum and Corynebacterium pyogenes with a combined bacterin. Onderstepoort Journal of Veterinary Research, 44 (4), 253--2;6 (1977). Rabbits were immunized with alum-precipitated, oil adjuvant and an untreated bacterin composed of F. necrophorum and C. pyogenes. Immunized rabbits were challenged intradermally with a mixture of F. necrophorum and C. pyrogenes. Immunized rabbits were challenged intradermally with a mixture of F. necorphorum and C. pyrogenes. Initially a low level of initial transient resistance could be demonstrated but a solid immunity could not be established.     

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.     

Swine dysentery: studies of gnotobiotic pigs inoculated with Treponema hyodysenteriae, Bacteroides vulgatus, and Fusobacterium necrophorum

Transmission experiments were carried out in gnotobiotic pigs to determine whether lesions typical of swine dysentery could be produced by oral inoculation of Treponema hyodysenteriae in combination with Bacteroides vulgatus or Fusobacterium necrophorum, or both. Each of the organisms had been isolated from swine with early lesions of the disease. Lesions were not found in 6 pigs inoculated with T hyodysenteriae alone, in 4 pigs given F necrophorum and T hyodysenteriae, or in 4 pigs given B vulgatus and F necrophorum. Lesions typical of swine dysentery developed in 8 pigs given B vulgatus, F necrophorum, and T hyodysenteriae as well as in 3 of 4 pigs given B vulgatus and T hyodysenteriae. In both of these groups, the inoculated bacteria were recovered from the colon, and T hyodysenteriae was demonstrated in the colonic crypts, epithelium, and lamina propria. The pathogenicity of the T hyodysenteriae was shown by the development of characteristic signs and lesions of swine dysentery in 12 of 14 naturally farrowed pigs inoculated with T hyodysenteriae alone.     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Pathogenicity of Fusobacterium necrophorum biovar C in mice by intraperitoneal and intraportal injections

Three strains of Fusobacterium necrophorum biovar C were injected into mice intraperitoneally and intraportally. All the mice survived. In one mouse out of 15 mice injected intraperitoneally, a few focal abscesses were formed in the liver. The microorganisms were recovered from the liver abscess and the tissue of liver with abscess. No changes were observed in the organs of other 14 mice and no bacteria were recovered from them. In the 15 mice injected intraportally, no liver abscesses and no macroscopic changes in the organs were formed. However, the inoculated bacteria were recovered from the liver of four mice. The pathogenicity of F. necrophorum biovar C was weaker than that of other two biovars.     

Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.     

Characterization of the 16S-23S rRNA intergenic spacer regions among strains of the Fusobacterium necrophorum cluster

The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.     

Effects of a collagenolytic cell wall component from Fusobacterium necrophorum subsp. necrophorum on rabbit tissue-culture cells

The effects on rabbit tissue-cultured cells of collagenolytic cell wall component (CCWC) from Fusobacterium necrophorum subsp. necrophorum were investigated. Scanning electron microscopy demonstrated that CCWC damaged the cell surfaces of the rabbit granulocytes and hepatocytes but the effects of the cells differed from each other. Granulocytes appeared smooth and morphologically irregular whereas hepatocytes looked rough and had tiny holes in the cell membranes. Differences in cell viability were observed in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt) assay. The findings suggest that cytotoxic activity in vivo may well contribute to the establishment of an initial injury in visceral tissues, and the action of CCWC could increase the chances of survival for an invading F. necrophorum subsp. necrophorum at the first stages of infection.