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J Med Microbiol 57 (2008), 225-231; DOI: 10.1099/jmm.0.47598-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity

Sambasivarao Tadepalli1,{dagger}, George C. Stewart2, T. G. Nagaraja1 and Sanjeev K. Narayanan1

1 Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, KS 66506-5606, USA

2 Department of Veterinary Pathobiology and Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA

Correspondence
Sanjeev K. Narayanan
sanjeev{at}vet.k-state.edu

Received 29 August 2007
Accepted 19 October 2007

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Abbreviations: PMN, polymorphonuclear leukocyte; PRAS, pre-reduced anaerobically sterilized.

{dagger}Present address: Novartis Animal Health US, Inc., Larchwood, IA 51241, USA. Back

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the human Fusobacterium necrophorum strains RMA10682 and RMA16505 reported in this paper are EF447426 and EF447427, respectively.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Fusobacterium necrophorum, a Gram-negative, rod-shaped obligate anaerobe, is a normal inhabitant of the gastrointestinal, respiratory and genitourinary tracts of animals and humans (Hagelskjaer & Prag, 2000; Nagaraja et al., 2005), and is associated with a variety of necrotic infections (Brazier et al., 2002; Navas et al., 1994; Razonable et al., 2003; Tan et al., 1996). In cattle, the organism is the primary causative agent of calf diphtheria, liver abscesses and foot rot (Nagaraja & Chengappa, 1998; Nagaraja et al., 2005; Tan et al., 1996). In humans, F. necrophorum primarily causes acute pharyngitis, thrombophlebitis and abscessation of the internal jugular vein, termed Lemierre's syndrome (Brazier, 2006; Hagelskjaer & Prag, 2000; Navas et al., 1994; Riordan & Wilson, 2004).

F. necrophorum has two major subspecies: subsp. necrophorum and subsp. funduliforme (Shinjo et al., 1991). In cattle, subsp. necrophorum is the most prevalent subspecies associated with infections and these strains generally produce significantly higher levels of leukotoxin than strains of subsp. funduliforme isolated from animals (Nagaraja et al., 2005; Scanlan et al., 1982; Tan et al., 1992). Based on biochemical tests, SDS-PAGE and pyrolysis MS, Hall et al. (1997) found that some human strains resembled animal subsp. funduliforme, whilst others resembled neither subspecies. Smith & Thornton (1997) also reported similar observations in their studies determining the pathogenic effects of animal and human strains in mice. Therefore, the synonymity of human strains with animal subsp. funduliforme cannot be established in the absence of 16S rRNA gene sequence data (Smith & Thornton, 1997).

Historically, F. necrophorum has been shown to be associated with Lemierre's syndrome in humans. However, recent studies indicate that it may also play an important role in persistent sore throat cases (Aliyu et al., 2004; Batty et al., 2005). Using real-time PCR methods, it has been reported that F. necrophorum strains isolated from persistent sore throat cases resembled subsp. funduliforme. However, in a recent study, Jensen et al. (2007) reported that F. necrophorum subsp. funduliforme is part of the normal flora of human tonsils. In view of these observations, it is essential to confirm the subspecies status of human strains by 16S rRNA gene sequencing.

In bovine F. necrophorum, production of leukotoxin is a major virulence factor (Narayanan et al., 2002a; Nagaraja et al., 2005). Leukotoxin is a large secreted protein that is cytotoxic to neutrophils and to a lesser extent to lymphocytes (Narayanan et al., 2001, 2002b). The leukotoxin operon (lktBAC) consists of three genes: lktB, lktA and lktC, of which lktA is the structural gene of leukotoxin (Narayanan et al., 2001; Oelke et al., 2005). The leukotoxin operon promoter-containing intergenic region varies in size and nucleotide sequence between the two subspecies (Zhang et al., 2006). Virulence factors implicated for human strains include the production of endotoxin and haemolysin (Brazier, 2006). Previous studies have shown that LPS from human strains resembles that of animal subsp. funduliforme in composition (being rich in amino sugars) (Brown et al., 1997). Studies to determine the biological activity of LPS have revealed that it is less toxic to rabbits (Hofstad & Kristoffersen, 1971). It is not known whether human F. necrophorum isolates have the lktA gene or leukotoxin activity. Our objectives were: (i) to subspeciate the human F. necrophorum strains based on their phenotypic and genotypic characteristics, and (ii) to determine whether human isolates contain the lktBAC operon and exhibit leukotoxic activity.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
F. necrophorum strains and subspeciation. Four human clinical isolates (RMA10682, RMA14786, RMA16505 and RMA16539) of F. necrophorum (kindly provided by Dr Diane Citron, R. M. Alden Research Laboratory, Santa Monica, CA, USA) were confirmed by growth, morphological and biochemical methods (Tan et al., 1994); the strains were isolated from a liver abscess, tonsil biopsy, tonsil swab and neck wound, respectively. Two bovine strains, F. necrophorum subsp. necrophorum A25 and subsp. funduliforme B35, previously isolated from bovine liver abscesses (Lechtenberg et al., 1988), were also included in the study. Isolates were grown overnight on blood agar (Remel) at 39 °C in an anaerobic glove box (Forma Scientific) and subcultured in pre-reduced anaerobically sterilized (PRAS) brain heart infusion (BHI) broth (Tan et al., 1992). Identification as F. necrophorum and subspeciation of the human strains were performed using a RapID ANA II system (Remel) and by PCR amplification of the rpoB gene (RNA polymerase β-subunit; Aliyu et al., 2004), haemagglutinin gene (Aliyu et al., 2004) and the leukotoxin operon promoter-containing intergenic region (Zhang et al., 2006). The partial 16S rRNA gene sequences of two human strains (RMA10682 and RMA16505), representative of the four, were determined using specific 16S forward and reverse primers (Table 1Go) that were designed based on the complete 16S rRNA gene sequence (GenBank accession no. AJ867038) of bovine F. necrophorum subsp. necrophorum. The obtained sequences were then aligned with the 16S rRNA gene sequences of 12 other species of the genus Fusobacterium using CLUSTAL_X (opening gap penalty of 10 and extension penalty of 0.1), and a phylogenetic tree (Dorsch et al., 2001) was constructed using a distance matrix algorithm and the neighbour-joining method using PAUP for Windows (Sinauer Associates).


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  		Table 1. Primers used in this study

 
Primers and PCR conditions. Specific primers targeting the rpoB and haemagglutinin genes have been designed previously (Aliyu et al., 2004). All primers used in this study are listed in Table 1Go. The leukotoxin operon promoter-containing intergenic regions in subsp. necrophorum and subsp. funduliforme were amplified using Ex Taq (Takara Bio) as described previously (Zhang et al., 2006). The PCR-amplified products were electrophoresed in 1–1.2 % agarose gels, purified and sequenced using a Beckman Coulter CEQ 8000 genetic analysis system.

Isolation of chromosomal DNA. Bacterial chromosomal DNA was prepared according to a procedure described previously (Narayanan et al., 2001). Briefly, isolates were grown overnight in anaerobic BHI broth. The cells were then pelleted by centrifugation at 5000 g for 10 min at 4 °C. The pelleted cells were washed and resuspended in 100 mM Tris/HCl (pH 8.0), 10 mM EDTA. The cell wall was digested with lysozyme (1 mg ml–1) and the resulting lysate was treated with 1 % sarkosyl followed by RNase A (20 µg ml–1) and Pronase (50 µg ml–1). The samples were incubated on ice for 20 min, and then sequentially extracted twice with phenol and chloroform. The DNA from the extract was precipitated in 0.1 vol. 3 M sodium acetate (pH 5.2) and an equal volume of 2-propanol. The DNA was resuspended in 10 mM Tris/HCl (pH 8.0), 1 mM EDTA and stored at 4 °C until use. The concentration of extracted DNA was determined spectrophotometrically (Nanodrop Technologies).

Southern blot hybridization. Southern blot hybridization was performed according to a procedure described previously (Narayanan et al., 2001). Briefly, 3 µg chromosomal DNA was digested to completion using HaeIII (Promega) and electrophoresed in a 1 % agarose gel overnight at 20 V at room temperature. The resolved and denatured DNA fragments were transferred to a positively charged nylon membrane (Roche Diagnostics) by a capillary mechanism, and the DNA immobilized by UV cross-linking. DNA probes were synthesized by random-labelling with DIG–dUTP (Roche Diagnostics) following the manufacturer's directions. The immobilized DNA was probed with cloned full-length bovine subsp. necrophorum A25 lktA at 54 °C (Narayanan et al., 2001), whilst the PCR fragments (produced in this study) of subsp. funduliforme B35 leukotoxin operon promoter-containing intergenic region (at 42 °C), subsp. funduliforme B35 lktB, subsp. necrophorum A25 lktB (at 50 °C) or subsp. necrophorum A25 lktC (at 44 °C) were used to probe the membrane. Colorimetric detection of hybridized signals was carried out following the manufacturer's instructions (Roche Diagnostics).

Preparation of culture supernatants. Bovine and human strains of F. necrophorum were grown in PRAS BHI to an OD600 value of 0.6–0.7 and pelleted by centrifugation at 5000 g for 20 min at 4 °C. The supernatant was collected, filtered through a 0.22 µm filter (Millipore) and concentrated 60-fold with 100 kDa molecular mass cut-off filters (Millipore). Aliquots were stored at –80 °C until use.

Western blotting. The concentrated culture supernatants from all strains were resolved by SDS-PAGE in 4–20 % Tris/glycine polyacrylamide gels (Pierce Biotechnology). Proteins were then electrotransferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 0.2 % skimmed milk overnight at 4 °C. Rabbit polyclonal antiserum raised against strain A25 native leukotoxin (Narayanan et al., 2001) was used to probe the membrane. Goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma-Aldrich) was used as the secondary antibody, and the immunoreactive proteins were detected using 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium as the substrate.

Preparation of white blood cells. Polymorphonuclear leukocytes (PMNs) from peripheral blood were prepared as described previously (Tan et al., 1992). Briefly, bovine whole blood was collected from healthy cattle by venipuncture of the internal jugular vein. The collected blood was transported on ice, centrifuged at 700 g for 10 min at 4 °C and the buffy coat removed to another sterile tube. The residual erythrocytes were subjected to osmotic lysis by sterile distilled water. Purified leukocytes were resuspended in complete RPMI 1640 containing 5 % fetal bovine serum, 100 IU penicillin l–1 and 100 mg streptomycin l–1 (Sigma-Aldrich). Human whole blood was collected by venipuncture from a healthy donor, erythrocytes were lysed with 6 vols RBC lysis buffer (Sigma-Aldrich) at room temperature for 5–10 min and the purified leukocytes were resuspended in complete RPMI 1640 (Oelke et al., 2005). The final concentration of viable cells was determined by a 0.4 % trypan blue dye exclusion assay (Narayanan et al., 2002b).

Cell viability assay. The cytotoxic effects of F. necrophorum culture supernatants were determined by cell viability assays using propidium iodide (Narayanan et al., 2002b). Briefly, 1x106 viable white blood cells were treated with culture supernatants for 45 min at 37 °C and 5 % CO2, washed twice and resuspended in 0.01 M PBS, and stained with 10 µl propidium iodide (50 µg ml–1 stock) in the dark for 5 min. White blood cells in complete RPMI 1640 treated similarly were used as a negative control. The samples were processed on a FACScan flow cytometer using an Argon ion laser (Becton Dickinson). Data were analysed using Cell Quest analysis software (Becton Dickinson).


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Subspeciation of F. necrophorum isolates

We employed growth, morphological and molecular methods to subspeciate the human strains of F. necrophorum. All four human strains were Gram-negative short rods, formed a flocculent button after overnight growth in PRAS BHI broth and were negative for alkaline phosphatase activity based on the RapID ANA II test. These results were indicative of the four strains belonging to subsp. funduliforme (Amoako et al., 1993; Tan et al., 1994). The rpoB gene (Fig. 1aGo) specific for F. necrophorum (Aliyu et al., 2004) was present in all four strains; however, all were PCR-negative for the haemagglutinin gene (Fig. 1bGo). The absence of the haemagglutinin gene was consistent with their subsp. funduliforme classification (Narongwanichgarn et al., 2003).


Figure 1
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  		Fig. 1. PCR amplification of the rpoB (RNA polymerase β subunit) gene (a), the haemagglutinin gene (b) and the leukotoxin operon promoter-containing intergenic region (c) from four human and two bovine strains of F. necrophorum. Lanes: M, 100 bp DNA ladder; 1, bovine F. necrophorum subsp. necrophorum strain A25; 2, bovine F. necrophorum subsp. funduliforme strain B35; 3–6, human F. necrophorum strains RMA10682, RMA14786, RMA16505 and RMA16539, respectively.

 
In cattle, the two subspecies of F. necrophorum can easily be distinguished by their growth, morphological and biochemical characteristics (Nagaraja et al., 2005). Previous studies to determine the subspecies of F. necrophorum human strains were inconclusive. Smith & Thornton (1993, 1997) attempted to classify human strains based on their pathogenic effects in mice. The pathogenic effects of most human strains resembled subsp. funduliforme, producing mild, localized lesions in mice, and none of them behaved like subsp. necrophorum. Hall et al. (1997) reported that human strains were clearly distinct from subsp. necrophorum of animal origin based on phenotypic reactions, pyrolysis MS and SDS-PAGE analysis, but similarity with subsp. funduliforme was less certain.

PCR amplification of the leukotoxin promoter-containing intergenic region of the human isolates revealed a subsp. funduliforme-type product (Fig. 1cGo). Results from Southern blot hybridization of HaeIII-digested genomic DNA probed with the DIG-labelled subsp. funduliforme leukotoxin operon promoter-containing intergenic region revealed that each of the human strains possessed a subsp. funduliforme-type promoter-containing intergenic region (Fig. 2aGo) and no signal was detected when probed with the labelled subsp. necrophorum promoter-containing intergenic region. Whilst the band size for strain RMA14786 was similar to that of bovine subsp. funduliforme (strain B35), strains RMA10682, RMA16505 and RMA16539 had smaller-sized bands. Nucleotide sequence analyses of the leukotoxin promoter-containing intergenic regions were identical to the subsp. funduliforme leukotoxin promoter-containing intergenic region (data not shown). Additionally, the aligned 16S rRNA gene sequences of the two strains were identical to each other and had greater identity (99 %) to subsp. funduliforme (data not shown). Therefore, the four human strains of F. necrophorum belonged to subsp. funduliforme.


Figure 2
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  		Fig. 2. Southern blot of HaeIII-digested genomic DNA hybridized with the DIG-labelled F. necrophorum subsp. funduliforme leukotoxin operon promoter-containing intergenic region (a), or with the F. necrophorum subsp. funduliforme lktB probe (b), lktA probe (c) or lktC probe (d). Lanes: M, DIG-labelled marker; 1, bovine F. necrophorum subsp. necrophorum strain A25; 2, bovine F. necrophorum subsp. funduliforme strain B35; 3–6, human F. necrophorum strains RMA10682, RMA14786, RMA16505 and RMA16539, respectively.

 
lktB gene

PCR analysis of human strains using primers of sequences conserved in the lktB gene yielded products of the expected size from all four strains. Nucleotide sequence analyses revealed greater sequence identity to subsp. funduliforme lktB than to subsp. necrophorum lktB. Southern blot hybridization with the subsp. necrophorum lktB probe resulted in a weak signal (data not shown) in comparison with the subsp. funduliforme lktB probe (Fig. 2bGo). Among the four human strains, there were differences in the size and intensity of the signals. Strains RMA14786 and B35 had similar-sized bands (~8.0 kb), whereas subsp. necrophorum lktB had a faintly hybridizing band of ~10.0 kb. Strains RMA10682, RMA16505 and RMA16539 had a smaller band of ~5.1 kb. A BLAST search of the partial lktB sequence of the human strains revealed 90–96 % identity with the bovine subsp. funduliforme lktB and 80–86 % identity with subsp. necrophorum lktB. The biological function of the LktB protein has not yet been determined.

lktA gene

The presence of the lktA gene was determined by digesting the genomic DNA to completion with HaeIII and probing with the subsp. necrophorum lktA in a Southern blot hybridization reaction (Fig. 2cGo). Genomic DNA digestion from subsp. necrophorum produced two hybridizing bands (~11 and ~10 kb) in Southern blot hybridizations, whereas subsp. funduliforme DNA yielded three bands (~9 kb, ~8 kb and a faint band of 375 bp). The bovine subsp. necrophorum lktA has a single recognition site (GenBank accession no. AF312861) for enzyme HaeIII, whereas subsp. funduliforme has two HaeIII sites (GenBank accession no. AY972049). Among the human strains, two unique hybridization patterns were observed. Strains RMA10682, RMA16505 and RMA16539 gave rise to a single strongly hybridizing band that varied in size from 8 to 9 kb, and a faintly hybridizing larger 10 kb band. Strain RMA14786 DNA gave rise to a doublet similar in size to that of subsp. funduliforme, but a larger hybridizing band similar to that observed in other human strains was also evident.

Thus, data from our Southern blot hybridization studies suggested that the strain RMA 14786 leukotoxin structural gene lktA may be more similar to subsp. funduliforme lktA, whereas, the lktA gene of the other three strains indicated fewer similarities to either subspecies. Instead of the doublet, a single band was observed in these three strains. This may be due to the absence of a recognition site for HaeIII in the proximity of the lktA gene in their genome. Hence, a single band of high molecular mass was observed in strains RMA16505 and RMA16539, and a distinct band equivalent to the lower-sized band in subsp. funduliforme was observed in strain RMA10682.

lktC gene

The lktC gene was identified by PCR using primers common to both subspecies. Sequencing of the PCR-amplified product revealed the presence of lktC in all four human strains. The sequencing results were further confirmed by Southern blot hybridization with a subsp. necrophorum lktC probe (Fig. 2dGo). The positive controls, bovine subsp. necrophorum and subsp. funduliforme, had the expected band sizes (~10 and ~7 kb, respectively). Two hybridization patterns were observed among the human strains. The first pattern (a band of ~1.5 kb) observed in strains RMA10682, RMA16505 and RMA16539 was different from either of the two bovine subspecies. The second pattern (a band of ~6.0 kb) was present only in strain RMA14786 and resembled the hybridization pattern of bovine subsp. funduliforme. The partial lktC nucleotide sequence of strain RMA14786 was similar (95 %) to the lktC sequence of both bovine subspecies. The lktC sequences of strains RMA10682, RMA16505 and RMA16539 resembled each other but were different from the two bovine subspecies. The biological function of the LktC protein has yet to be determined.

Western blotting

The leukotoxin from F. necrophorum is highly unstable and breaks down into multiple products under denaturing conditions (Tan et al., 1994; Fig. 3Go). Culture supernatant from subsp. necrophorum had all the expected intense bands (~250, 150, 130 and 110 kDa) (Fig. 3Go, lane 1), whereas the supernatant from subsp. funduliforme produced less-intense bands (Fig. 3Go, lane 2). Two patterns of hybridization were observed in the culture supernatants of human clinical isolates. The first hybridization pattern seen in the culture supernatant of strain RMA14786 resembled that of subsp. funduliforme with fewer bands and a weaker affinity. The other three strains gave rise to variably intense bands of 150 and 40 kDa, which were similar to each other but distinct from that of subsp. necrophorum.


Figure 3
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  		Fig. 3. Western blot of concentrated culture supernatants probed with rabbit polyclonal serum raised against the LktA protein from bovine F. necrophorum subsp. necrophorum. Lanes: M, pre-stained protein marker; 1, bovine F. necrophorum subsp. necrophorum strain A25; 2, bovine F. necrophorum subsp. funduliforme strain B35; 3–6, human strains RMA10682, RMA14786, RMA16505 and RMA16539, respectively.

 
Leukotoxin activity

Viability assays using human peripheral PMNs indicated that all of the culture supernatants from the human strains were toxic (Fig. 4Go); subsp. necrophorum and funduliforme culture supernatants were also toxic to human PMNs. In order to confirm that the observed toxicities were not due to contaminating LPS, we tested the toxic effects of the culture supernatants after passing them over a polymyxin B column. No significant decrease in toxic activity was observed (data not shown). Similar strain-to-strain variations in the cytotoxic effects of leukotoxin among the bovine strains of F. necrophorum have been reported previously (Tan et al., 1992). Significant decreases in leukotoxin activity were observed when culture supernatants were pre-treated with the proteolytic enzymes trypsin, chymotrypsin and protease (Coyle-Dennis & Lauerman, 1978, 1979; Tan et al., 1994). Hence, the differences in cytotoxic activity among human strains may be related to the various proteolytic enzyme(s) secreted by individual strains, or to yet-to-be identified factors.


Figure 4
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  		Fig. 4. Cell viability assay. Human PMNs were treated with culture supernatants at 37 °C and 5 % CO2 for 45 min, washed and then incubated with propidium iodide. PMNs were analysed by flow cytometry and 10 000 events were recorded. Culture supernatants: 1, RPMI 1640 only (no toxin); 2, bovine F. necrophorum subsp. necrophorum strain A25; 3, bovine F. necrophorum subsp. funduliforme strain B35; 4–7, human F. necrophorum strains RMA10682, RMA14786, RMA16505 and RMA16539, respectively. Results are shown as means±SD of three independent assays.

 
Conclusions

In conclusion, our study showed that the four human strains belonged to subsp. funduliforme and contained the leukotoxin operon lktBAC. The secreted leukotoxin was toxic to human PMNs. It is possible that leukotoxin may be an important virulence factor and may have a role in human F. necrophorum infections. Further research on the role of the leukotoxin of F. necrophorum is warranted.


   ACKNOWLEDGEMENTS
 
We thank David George for DNA sequencing, Tammy Koopman for assistance with the flow cytometer and Ashvin Nagaraja for help in the laboratory. This paper is contribution no.08-00-j from the Kansas Agricultural Experiment Station.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Aliyu, S. H., Marriott, R. K., Curran, M. D., Parmar, S., Bentley, N., Brown, N. M., Brazier, J. S. & Ludlam, H. (2004). Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice. J Med Microbiol 53, 1029–1035.[Abstract/Free Full Text]

Amoako, K. K., Goto, Y. & Shinjo, T. (1993). Comparison of extracellular enzyme of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme. J Clin Microbiol 31, 2244–2247.[Abstract/Free Full Text]

Batty, A., Wren, M. W. D. & Gal, M. (2005). Fusobacterium necrophorum as the cause of recurrent sore throat: comparison of isolates from persistent sore throat syndrome and Lemierre's disease. J Infect 51, 299–306.[CrossRef][Medline]

Brazier, J. S. (2006). Human infections with Fusobacterium necrophorum. Anaerobe 12, 165–172.[CrossRef][Medline]

Brazier, J. S., Hall, V., Yusuf, E. & Duerden, B. I. (2002). Fusobacterium necrophorum infections in England and Wales 1990–2000. J Med Microbiol 51, 269–272.[Abstract/Free Full Text]

Brown, R., Lough, H. G. & Poxton, I. R. (1997). Phenotypic characteristics and lipopolysaccharides of human and animal isolates of Fusobacterium necrophorum. J Med Microbiol 46, 873–878.[Abstract/Free Full Text]

Coyle-Dennis, J. E. & Lauerman, L. H. (1978). Biological and biochemical characteristics of Fusobacterium necrophorum leukotoxin. Am J Vet Res 39, 1790–1793.[Medline]

Coyle-Dennis, J. E. & Lauerman, L. H. (1979). Correlations between leukocidin production and virulence of two isolates of Fusobacterium necrophorum. Am J Vet Res 40, 274–276.[Medline]

Dorsch, M., Love, D. N. & Bailey, G. D. (2001). Fusobacterium equinum sp. nov., from the oral cavity of horses. Int J Syst Evol Microbiol 51, 1959–1963.[Abstract]

Hagelskjaer, K. L. & Prag, J. (2000). Human necrobacillosis, with emphasis on Lemierre's syndrome. Clin Infect Dis 31, 524–532.[CrossRef][Medline]

Hall, V., Duerden, B. I., Magee, J. T., Ryley, H. C. & Brazier, J. S. (1997). A comparative study of Fusobacterium necrophorum strains from human and animal sources by phenotypic reactions, pyrolysis mass spectrometry and SDS-PAGE. J Med Microbiol 46, 865–871.[Abstract/Free Full Text]

Hofstad, T. & Kristoffersen, T. (1971). Preparation and chemical characteristics of endotoxic lipopolysaccharide from three strains of Sphaerophorus necrophorus. . Acta Pathol Microbiol Scand B Microbiol Immunol 79, 385–390.[Medline]

Jensen, A., Hagelskjaer Kristensen, L. & Prag, J. (2007). Detection of Fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time PCR. Clin Microbiol Infect 13, 695–701.[CrossRef][Medline]

Lechtenberg, K. F., Nagaraja, T. G., Leipold, H. W. & Chengappa, M. M. (1988). Bacteriological and histological studies of hepatic abscesses in cattle. Am J Vet Res 49, 58–62.[Medline]

Nagaraja, T. G. & Chengappa, M. M. (1998). Liver abscesses in feedlot cattle: a review. J Anim Sci 76, 287–298.[Abstract/Free Full Text]

Nagaraja, T. G., Narayanan, S. K., Stewart, G. C. & Chengappa, M. M. (2005). Fusobacterium necrophorum infections in animals: pathogenesis and pathogenic mechanisms. Anaerobe 11, 239–246.[CrossRef][Medline]

Narayanan, S. K., Nagaraja, T. G., Chengappa, M. M. & Stewart, G. C. (2001). Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum. Infect Immun 69, 5447–5455.[Abstract/Free Full Text]

Narayanan, S. K., Nagaraja, T. G., Chengappa, M. M. & Stewart, G. C. (2002a). Leukotoxins of gram-negative bacteria. Vet Microbiol 84, 337–356.[CrossRef][Medline]

Narayanan, S. K., Stewart, G. C., Chengappa, M. M., Willard, L., Shuman, W., Wilkerson, M. & Nagaraja, T. G. (2002b). Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes. Infect Immun 70, 4609–4620.[Abstract/Free Full Text]

Narongwanichgarn, W., Misawa, N., Jin, J. H., Amoako, K. K., Kawaguchi, E., Shinjo, T., Haga, T. & Goto, Y. (2003). Specific detection and differentiation of two subspecies of Fusobacterium necrophorum by PCR. Vet Microbiol 91, 183–195.[CrossRef][Medline]

Navas, E., Martinez-San, J. M., Garcia-Villanueva, M. & de Blas, A. (1994). Brain abscess with intracranial gas formation: case report. Clin Infect Dis 19, 219–220.[Medline]

Oelke, A. M., Nagaraja, T. G., Wilkerson, M. J. & Stewart, G. C. (2005). The leukotoxin operon of Fusobacterium necrophorum is not present in other species of Fusobacterium. Anaerobe 11, 123–129.[CrossRef][Medline]

Razonable, R. R., Rahman, A. E. & Wilson, W. R. (2003). Lemierre syndrome variant: necrobacillosis associated with inferior vena cava thrombosis and pulmonary abscesses after trauma-induced leg abscesses. Mayo Clin Proc 78, 1153–1156.[Abstract/Free Full Text]

Riordan, T. & Wilson, M. (2004). Lemierre's syndrome: more than a historical curiosa. Postgrad Med J 80, 328–334.[Abstract/Free Full Text]

Scanlan, C. M., Berg, J. N. & Fales, W. H. (1982). Comparative in vitro leukotoxin production of three bovine strains of Fusobacterium necrophorum. Am J Vet Res 43, 1329–1333.[Medline]

Shinjo, T., Fujisawa, T. & Mitsuoka, T. (1991). Proposal of two subspecies of Fusobacterium necrophorum (Flugge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flugge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Halle 1898). Int J Syst Bacteriol 41, 395–397.[Abstract/Free Full Text]

Smith, G. R. & Thornton, E. A. (1993). Pathogenicity of Fusobacterium necrophorum strains from man and animals. Epidemiol Infect 110, 499–506.[Medline]

Smith, G. R. & Thornton, E. A. (1997). Classification of human and animal strains of Fusobacterium necrophorum by their pathogenic effects in mice. J Med Microbiol 46, 879–882.[Abstract/Free Full Text]

Tan, Z. L., Nagaraja, T. G. & Chengappa, M. M. (1992). Factors affecting the leukotoxin activity of Fusobacterium necrophorum. Vet Microbiol 32, 15–28.[CrossRef][Medline]

Tan, Z. L., Nagaraja, T. G. & Chengappa, M. M. (1994). Biochemical and biological characterization of ruminal Fusobacterium necrophorum. FEMS Microbiol Lett 120, 81–86.[CrossRef][Medline]

Tan, Z. L., Nagaraja, T. G. & Chengappa, M. M. (1996). Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures. Vet Res Commun 20, 113–140.[CrossRef][Medline]

Zhang, F., Nagaraja, T. G., George, D. & Stewart, G. C. (2006). The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions. Vet Microbiol 112, 73–78.[CrossRef][Medline]




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H. A. Ludlam, N. J. Milner, J. S. Brazier, I. H. Davies, K. Perry, R. K. Marriott, L. Donachie, and M. D. Curran
lktA-encoded leukotoxin is not a universal virulence factor in invasive Fusobacterium necrophorum infections in animals and man
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