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transfer of kingella indologenes (snell and lapage 1976) to the genus suttonella gen. nov. as suttonella indologenes comb. nov.; transfer of bacteroides nodosus (beveridge 1941) to the genus dichelobacter gen. nov. as dichelobacter nodosus comb. nov.; and assignment of the genera cardiobacterium, dichelobacter, and suttonella to cardiobacteriaceae fam. nov. in the gamma division of proteobacteria on the basis of 16s rrna sequence comparisons.the 16s rrna sequences of kingella indologenes, cardiobacterium hominis, and bacteroides nodosus were determined by direct rna sequencing, using a modified sanger method. sequence comparisons indicated that these three species represent a novel family in the gamma division of proteobacteria. on the basis of these data, k. indologenes and b. nodosus cannot retain their current generic status as they are not closely related to other members of their assigned genera. therefore, we propose transfer ...19902275858
sequence of fimbrial subunit-encoding genes from virulent and benign isolates of dichelobacter (bacteroides) nodosus.both virulent and benign isolates of the ovine pathogen dichelobacter (bacteroides) nodosus produce polar fimbriae which have been implicated in twitching motility. the fimbrial subunit-encoding genes from two virulent and two benign serogroup-b isolates of d. nodosus were cloned and sequenced. analysis of the deduced amino acid (aa) sequences of these subunits indicated the presence of substitutions that appeared to correlate with the virulence phenotype. however, these aa substitutions were lo ...19911673665
shared antigenicity and immunogenicity of type 4 pilins expressed by pseudomonas aeruginosa, moraxella bovis, neisseria gonorrhoaea, dichelobacter nodosus, and vibrio cholerae.immunoblotting with polyclonal rabbit antibodies raised against pilins expressed by pseudomonas aeruginosa, moraxella bovis, neisseria gonorrhoeae, dichelobacter nodosus, and vibrio cholerae was used to demonstrate that these polypeptides display conserved antigenic and, in most cases, immunogenic determinants. these determinants appear to be localized to the highly homologous amino-terminal domains (residues 1 to 25).19911682267
identification of three gene regions associated with virulence in dichelobacter nodosus, the causative agent of ovine footrot.dichelobacter nodosus (formerly bacteroides nodosus) is a gram-negative strict anaerobe and is the primary pathogen involved in ovine footrot. a comparative hybridization strategy was used to isolate recombinant clones which hybridized to dna from a virulent strain of d. nodosus but not with a benign isolate. three virulence-associated gene regions were identified and one of these regions was shown to be present in multiple copies in the d. nodosus genome. hybridization studies on 101 clinical i ...19911748867
the type 4 pilin of moraxella nonliquefaciens exhibits unique similarities with the pilins of neisseria gonorrhoeae and dichelobacter (bacteroides) nodosus.moraxella nonliquefaciens is a bacterium which is part of the normal flora of the human upper respiratory tract and is an occasional cause of disease. using a previously cloned type 4 pilin gene (tfpq) from moraxella bovis as a hybridization probe, we have cloned an 826 bp sau3 ai fragment which contains an m. nonliquefaciens type 4 pilin gene (tfpa) from strain nctc 7784. the pilin gene is expressed in escherichia coli. we have examined nctc 7784 and nine other m. nonliquefaciens strains by gen ...19911770363
humoral responses to a multivalent vaccine in age-matched lambs of different bodyweight and nutrition.k-agglutination, pilus-enzyme-linked immunosorbent assay (elisa) and outer membrane protein-elisas were used to assess humoral responses after vaccination with a commercial, multivalent, ovine foot rot vaccine (dichelobacter nodosus whole cells) in three groups of nine-month-old lambs of markedly different bodyweight, nutritional history and dietary protein supply. mean bodyweights of lambs in low (l), medium (m) and high (h) bodyweight/nutrition groups were 22, 32 and 48 kg, respectively, at th ...19921352408
molecular characterization of a genomic region associated with virulence in dichelobacter nodosus.the major pathogen implicated in footrot, a highly contagious disease of sheep, is the strict anaerobe dichelobacter nodosus (formerly bacteroides nodosus). sequence analysis of a 2,262-bp segment of the d. nodosus genome which is more prevalent in virulent isolates than in other isolates showed the presence of four open reading frames which appeared to have consensus transcriptional and translational start signals. these virulence-associated genes have been designated vapabcd. two of the three ...19921398971
amino acid and dna sequences of an extracellular basic protease of dichelobacter nodosus show that it is a member of the subtilisin family of proteases.a dna fragment encoding an extracellular basic protease (pi approximately 9.5) from dichelobacter nodosus, a gram-negative obligate anaerobe and the causative agent of ovine footrot, has been cloned and expressed in escherichia coli and sequenced. e. coli harbouring a plasmid with a 3-kb dna fragment containing the d. nodosus basic-protease gene exhibited proteolytic activity when tested on skim-milk plates. the sequence of the native basic protease isolated from d. nodosus was also determined b ...19921446666
evaluation of a simple method for improving the precision of an elisa detecting antibody in serum.a method of correcting od values to improve between-assay precision (pcf4 method) was evaluated using an elisa detecting anti-dichelobacter nodosus antibody in ovine serum. four control sera with pre-defined target od were included on each plate and used to calculate a correction factor. the mean between-plate cv for eight reference sera tested over a period of 6 months was reduced from 15.2% to 9.4% in a polyclonal conjugate assay (114 plates) and from 13.3% to 9.8% in a monoclonal conjugate as ...19921564335
amino acid sequence of extracellular acidic protease v5 of dichelobacter nodosus, the causative organism of ovine footrot.dichelobacter nodosus, a gram-negative obligate anaerobe and the causative organism of ovine footrot, secretes a family of extracellular serine proteases with pi's in the range of 5.2 to 5.6 and a serine basic protease with a pi of approximately 9.5. the primary structure of acidic protease v5 (pi approximately 5.2) from d. nodosus virulent strain 198 was determined by direct amino acid sequencing. this protease consists of a single polypeptide chain of 347 amino acids, contains two disulfide bo ...19938330022
a gelatin test to detect activity and stability of proteases produced by dichelobacter (bacteroides) nodosus.previously reported tests to distinguish thermostable and thermolabile proteases of dichelobacter nodosus used hide powder azure as a test substrate. this paper describes an alternative test for protease stability using gelatin, an inexpensive and convenient substrate. the test required less equipment and time than the hide powder tests, and simplified the testing of multiple samples. proteases from 2965 isolates of d. nodosus from samples collected as part of a footrot eradication scheme were t ...19938236773
dichelobacter nodosus: differentiation of virulent and benign strains by gene probe based dot blot hybridisation.gene probes specific for benign and virulent strains of dichelobacter nodosus were used in a dot blot hybridisation procedure involving 96 strains of d. nodosus isolated from cases of ovine footrot. the performance of the probes was compared with that of the elastase test. all 27 strains with elastase activity at 7 days and 12 of 25 strains with elastase activity at 14 days reacted with the virulent-specific probe. twenty-four strains with elastase activity between 21-28 days, and 20 strains wit ...19938128604
the effects of antigenic competition on the efficacy of multivalent footrot vaccines.a multivalent footrot vaccine has been developed, containing pilus antigens produced in recombinant pseudomonas aeruginosa and representing all nine serogroups of dichelobacter (bacteroides) nodosus commonly recognised in the field. the responses of sheep to the multivalent vaccine have been compared with those to monovalent vaccines representing only a single serogroup. antigenic competition between serogroups occurred in sheep immunised with the multivalent formation, but high levels of protec ...19938098601
recombinant vaccines against ovine footrot.for the past 20 years footrot vaccines have evolved from simple bacterins to highly specific recombinant dna (rdna) fimbrial vaccines. the development of these vaccines has left a trail of discoveries, challenges and solutions; these processes continue as we move closer to understanding the requirements of a footrot vaccine. the initial whole cell vaccines were unsuccessful due to the short duration of immunity and incorporation of limited serotypes. a multistrain vaccine eliminated the problem ...19937505770
cloning, sequence and expression of the gene (aprv5) encoding extracellular serine acidic protease v5 from dichelobacter nodosus.the acidic protease v5-encoding gene (aprv5) from gram- dichelobacter nodosus virulent strain 198 was isolated from a cosmid bank by activity screening and sequenced. the 2371-bp nucleotide (nt) sequence contained an open reading frame coding for a protein precursor of 595 amino acid (aa) residues composed of a signal peptide, a pro-region, a mature active protease of 347 aa and a c-terminal extension region of 120 aa. the deduced aa sequence of the pre-pro-mature protease regions showed about 6 ...19938299956
detection of dichelobacter nodosus using species-specific oligonucleotides as pcr primers.dichelobacter nodosus is an essential causative agent of ovine footrot, a disease of major economic significance. four oligonucleotides complementary to variable regions of the 16s rrna of d. nodosus were identified, synthesized and tested for their specificity and sensitivity as probes for the detection of d. nodosus. in hybridization reactions using total rna as the target nucleic acid, three probes were found to be both sensitive and species-specific. when these probes were used as primers in ...19937689774
use of a gram- signal peptide for protein secretion by gram+ hosts: basic protease of dichelobacter nodosus is produced and secreted by bacillus subtilis.the bprv gene, encoding the extracellular basic protease of the gram- anaerobic bacterium dichelobacter nodosus, was expressed and the protein secreted in bacillus subtilis using the novel cloning/expression vector pnc3 [wu et al., gene 106 (1991) 103-107]. the pre- and pro-peptides were processed correctly in this heterologous system, and the 127-amino acid c-terminal extension region was also removed. the recombinant gene product was indistinguishable biochemically or immunochemically from the ...19938370546
use of elastase test, gelatin gel test and electrophoretic zymogram to determine virulence of dichelobacter nodosus isolated from ovine foot rot.a comparative study using the elastase test, gelatin gel test and electrophoretic zymogram was conducted for virulence determination of 96 dichelobacter nodosus isolates from clinical cases of ovine foot rot. despite being time consuming, the elastase test provided a classification of d nodosus bacteria in general agreement with the severity of clinical characteristics. the gelatin gel test showed some correlation with the elastase test. while isolates showing elastase activity at 14 days or les ...19938378606
benign footrot--an epidemiological investigation into the occurrence, effects on production, response to treatment and influence of environmental factors.benign footrot was studied in 1 1/2-years-old merinos on 2 farms in central victoria from september 1987 to august 1990, inclusive. treatment groups of 100 sheep grazed together with the remaining untreated sheep. inspections were carried out every 3 weeks during the spring transmission period until the number of lesions greater than score 2 dropped below 3%. at each inspection, each sheep was weighed and lesion scores for each foot and digit were recorded, the treated group of sheep was treated ...19938460991
properties of the extracellular acidic proteases of dichelobacter nodosus. stability and specificity of peptide bond cleavage.dichelobacter nodosus, a gram negative obligate anaerobe and causative organism of ovine footrot, secretes a family of extracellular acidic serine proteases with pi's in the range of 5.2 to 5.6, and a basic serine protease with a pi of approximately 9.5. the acidic proteases show optimum activity at ph 8 and require a divalent metal ion (eg. ca) to maintain structural integrity. in the presence of edta or conditions that cause protein unfolding, the proteases undergo rapid and complete autolysis ...19947696989
characterization of the serine protease and serine protease inhibitor from the tissue-penetrating nematode anisakis simplex.a serine protease and a serine protease inhibitor were purified from infective larvae of the parasitic nematode anisakis simplex. the serine protease was found to be trypsin-like and preferentially cleaved substrates with the basic amino acid arginine at the p1 position (z-gly-pro-arg-amc (where z is benzyloxycarbonyl), km = 0.019 mm, and z-phe-pro-arg-amc, km = 0.013 mm) at rates similar to those determined for trypsin (0.002 mm and 0.006 mm, respectively). however, the presence of a bulky hydr ...19947961683
development of gene probes of dichelobacter nodosus for differentiating strains causing virulent, intermediate or benign ovine footrot.seven dichelobacter nodosus genomic dna clones including six specific for virulent and one for benign strains were identified. a collection of 96 footrot isolates, which in turn comprised 27 virulent isolates showing elastase activity at 7 days, 25 high intermediate isolates with elastase activity at 14 days, 24 low intermediate isolates with elastase activity at 21-28 days and 20 benign isolates with no elastase activity at up to 28 days, were used to assess these clones. of the six virulent sp ...19947953579
antigenic competition in a multivalent foot rot vaccine.the antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. our prototype vaccine contains pili from nine serogroups of dichelobacter nodosus which are expressed in pseudomonas aeruginosa. sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. levels of agglutinating and total antibody specific f ...19947912871
the laboratory culture of dichelobacter nodosus in a footrot eradication program.as part of a program to eradicate virulent footrot from western australia, 2745 isolates of dichelobacter nodosus were isolated from 5263 specimens from 1883 submissions. the virulence of each isolate was assessed using protease thermostability and isoenzyme zymogram. we describe changes to the materials and methods required to handle these specimens and to reduce the interval between submission and report to 8 days.19948048905
sequencing and expression of the aroa gene from dichelobacter nodosus.the aroa locus of the gram- pathogen dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (epsp) synthase, has been sequenced and expressed in escherichia coli. the gene is located on a 1.48-kb drai-hindiii fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. the deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated m(r) of 47,41 ...19948045432
experimental evaluation of a commercial footrot vaccine against native canadian strains of dichelobacter nodosus.two serotypes of the anaerobic bacterium dichelobacter nodosus were used to experimentally infect young sheep resulting in infectious pododermatitis or footrot characteristic of the natural disease in sheep. the specific serotypes of d. nodosus were reisolated from the feet and identified using immunofluorescent microscopy of hoof scrapings. prior immunization of sheep with a commercially available bacterin containing whole cell preparations of ten strains of d. nodosus resulted in serum igg rea ...19948004537
expression in escherichia coli of the extracellular basic protease from dichelobacter nodosus.dichelobacter nodosus, a gram-negative obligate anaerobe and the causative agent of ovine footrot, secretes a number of extracellular proteases, one of which is highly basic in nature. the gene (bprv) encoding this basic protease, from virulent strain 198, has been cloned and sequenced. clone pbr3kb contained the complete bprv gene which constitutively expressed an active protease using its own promoter, when cloned in escherichia coli. however, levels of protease expression were low and unstabl ...19947921259
protective antibody titres and antigenic competition in multivalent dichelobacter nodosus fimbrial vaccines using characterised rdna antigens.the relationship between k-agglutination antibody titres and protection against experimental challenge with dichelobacter nodosus, the effect of increasing the number of d. nodosus fimbrial antigens, and the importance of the nature of additional antigens in multivalent vaccines on antibody response and protection against experimental challenge with d. nodosus were examined in merino sheep. a total of 204 merino sheep were allocated to one of 12 groups, and vaccinated with preparations containin ...19947909183
double immunogold labelling demonstrating expression of recombinant genes for production of an anti-fertility vaccine.the multiple antibody technique for double immunogold labelling for the simultaneous localization of two antigens with negative staining was utilized to demonstrate the expression of recombinant genes in bacteria, with the primary antibodies being raised in different host species. for the production of a vaccine for immunological control of fertility, a multi-functional plasmid vector was introduced into the bacterium pseudomonas aeruginosa containing the dichelobacter nodosus fimbrial subunit g ...19947881895
application of elisa to the serological diagnosis of virulent ovine footrot.the sensitivity and specificity of an elisa that detects antibody in the serum of sheep against dichelobacter nodosus was investigated. the sensitivity of the assay was determined in two trials in which sheep free of virulent footrot were exposed to a virulent strain of d. nodosus. in the first trial up to 96.6% of sheep that developed severe foot lesions were seropositive 2 weeks later. the proportion of seropositive sheep depended on the interval between exposure to d. nodosus and collection o ...19947801518
characterization of a basic serine proteinase (pi approximately 9.5) secreted by virulent strains of dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains.an extracellular serine proteinase with a pi approximately 9.5 (referred to as 'basic proteinase') was purified to homogeneity, from strains of dichelobacter nodosus that cause virulent foot-rot, by gel filtration of concentrated culture supernatant on sephadex g-100 and chromatography on sulphopropyl-sephadex c-25 at ph 8.6 d. nodosus strains that cause benign foot-rot do not secrete a corresponding basic proteinase with a pi of approximately 9.5. benign strains secrete a closely related, but d ...19948172614
genetic organization of the duplicated vap region of the dichelobacter nodosus genome.the recombinant plasmid pjir318 contains a fragment of the dichelobacter nodosus genome which is associated with virulence. sequence analysis of the pjir318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the escherichia coli f plasmid and the neisseria gonorrhoeae cryptic plasmid (m. e. katz, r. a. strugnell, and j. i. rood, infect. and immun. 60:4586-4592, 1992). the plasmid pjir318 hybridizes to three regions ...19948169216
purification of the extracellular acidic proteases of dichelobacter nodosus.dichelobacter nodosus, a gram negative obligate anaerobe and causative organism of ovine footrot, secretes a family of extracellular acidic serine proteases with pi's in the range of 5.2 to 5.6, and a basic serine protease with a pi of approximately 9.5. four acidic proteases (v1, v2, v3 and v5) from virulent and five acidic proteases (b1 to b5) from benign strains of d. nodosus were purified by chromatography on sephadex g-100 and deae-sepharose cl-6b. proteases v2, v5 and b5 were found to yiel ...19947696988
a retrospective study of clinical and laboratory characteristics of ovine footrot.the infection of the feet of sheep by the anaerobic bacterium dichelobacter nodosus results in a spectrum of diseases ranging from virulent through intermediate to benign footrot. by examining the clinical characteristics of various forms of footrot from 22 properties in victoria, australia in association with the results of several laboratory tests including the gene probe-based dot blot hybridisation, it was shown that virulent footrot could not be differentiated from intermediate footrot usin ...19949133062
disease resistance in merino sheep. iii. genetic variation in resistance to footrot following challenge and subsequent vaccination with an homologous rdna pilus vaccine under both induced and natural conditions.summary: eight traits representing clinical indicators of resistance to footrot were examined in 1562 merino sheep, representing the progeny from 162 sires in four major bloodlines. over a 4-year period, sheep were exposed to virulent isolates of dicbelobacter nodosus under both an experimental challenge in which footrot was induced, and a separate natural challenge involving a different isolate of d. nodosus. five footrot traits and three healing traits were each recorded on seven occasions fol ...199421395787
a multiple site-specific dna-inversion model for the control of omp1 phase and antigenic variation in dichelobacter nodosus.the molecular cloning and sequence analysis of four structurally variant linked genes (omp1a,b,c,d) that encode the major outer membrane protein of dichelobacter nodosus strain vcs1001 are described. the isolation of rearranged copies of omp1a and omp1b, and the identification in the 5' regions of all four genes of short cross-over-site sequences that were similar to the din family of cross-over-site sequences, suggested that site-specific dna inversion was involved in omp1 rearrangement. eviden ...19957476204
delineation of the virulence-related locus (vrl) of dichelobacter nodosus.dichelobacter nodosus is the primary pathogen implicated in ovine footrot. in this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent d. nodosus isolates. the precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain a198 were compared to corresponding sequences from the benign isolate c305. the left end of the vrl locus was located in a sequence similar to that of the small ...19957496519
a gene region in dichelobacter nodosus encoding a lipopolysaccharide epitope.dichelobacter nodosus is a gram-negative anaerobic bacterium that is the causative organism of footrot in sheep. a d. nodosus locus responsible for a modification of the host lipopolysaccharide (lps) in escherichia coli was cloned and sequenced. genetic studies showed that the modification occurred within the inner-core region of the host lps, most likely to one or more of the heptose molecules. antibodies eluted from the modified lps reacted preferentially with the lipid-a-core region of d. nod ...19957539688
identification of fimbrial assembly genes from dichelobacter nodosus: evidence that fimp encodes the type-iv prepilin peptidase.dichelobacter nodosus (dn) is the causative agent of footrot, an economically significant disease of sheep. one of the factors believed to be involved in the virulence of this organism is its ability to produce type-iv fimbriae, which are the major protective antigens. to investigate the process of fimbrial biogenesis in dn, gene probes were constructed from pilus biogenesis genes of pseudomonas aeruginosa (pa) and used to isolate homologues from dn. a homologue, designated fimp, of the pa prepi ...19957642131
a polymerase chain reaction assay for improved determination of virulence of dichelobacter nodosus, the specific causative pathogen for ovine footrot.three sets of oligonucleotide primers (vf1 and vr1, vf2 and vr2, bf and br) were derived from a d. nodosus virulent-specific clone pv470-13 (2146 bp) and a benign-specific clone pb645-335 (737 bp), respectively. using the virulent-specific primers vf1 and vr1 in a polymerase chain reaction (pcr) enabled amplification of a dna fragment of 460 bp in 25/27 virulent, 9/25 high intermediate, 9/24 low intermediate and 2/20 benign isolates of d. nodosus. on the other hand, using the second set of the v ...19957740758
expression and secretion of heterologous proteases by corynebacterium glutamicum.genes encoding the basic protease of dichelobacter nodosus (bprv) and the subtilisin of bacillus subtilis (apre) were cloned and expressed in corynebacterium glutamicum. in each case, enzymatically active protein was detected in the supernatants of liquid cultures. while the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprv basic protease did not facilitate secretion. a hybrid apre-bprv gene in which the promoter and signal peptide coding sequ ...19957747974
nucleotide and deduced protein sequence of the extracellular, serine basic protease gene (bprb) from dichelobacter nodosus strain 305: comparison with the basic protease gene (bprv) from virulent strain 198.in earlier studies, it appeared that benign strains of the gram-negative, obligate anaerobe, dichelobacter nodosus, were devoid of the extracellular, serine basic protease (pi approximately 9.5) of virulent strains. however, southern and pcr analysis have shown a homologous gene (bprb) in the representative benign strain 305. the deduced amino acid sequence of the prepro- and mature protease regions of bprb confirmed this homology and showed 97% sequence identity with the bprv precursor from vir ...19957663404
observations on the indirect transmission of virulent ovine footrot in sheep yards and its spread in sheep on unimproved pasture.virulent ovine footrot was transmitted accidentally to a group of 23 adult merino sheep (flock b) after holding for 1 hour in sheep yards, which earlier the same day had contained another flock (flock a) with < 1% prevalence of sheep with footrot lesions. sheep in flock b were rendered susceptible to virulent footrot by grazing 600 mm high unimproved pasture dominated by paspalum (paspalum dilatatum) and kangaroo grass (themeda australis) during warm, humid and wet weather. in addition to moistu ...19957646377
a role for bacteriophages in the evolution and transfer of bacterial virulence determinants.a virulence-associated region in the genome of dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of shigella flexneri phage sf6 and coliphages p4 and phi r73, together with open reading frames (vapb, c and d) related to genes borne on plasmids in neisseria gonorrhoeae, escherichia coli, actinobacillus actinomycetemcomitans and treponema denticola. similar to p4 and phi r73, the vap region is bracketed by putative bacteriophage att sites a ...19958709840
molecular basis for the virulence of dichelobacter nodosus. 19958800839
a single amino-acid change between the antigenically different extracellular serine proteases v2 and b2 from dichelobacter nodosus.dichelobacter nodosus (dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease v5 and a basic protease have already been characterised from virulent dn strain 198. the gene encoding the third protease type, as represented by acidic protease v2, was isolated from an ecori-bamhi l ...19958566792
grading the lesions of ovine footrot.sixteen methods of grading the lesions of ovine footrot were assessed on the basis of the effect of the lesions on the humoral immune response of the host to a causative bacterium, dichelobacter nodosus. methods that allowed for qualitative and quantitative differences in lesion scores between sheep were the best predictors of host response, and methods that assessed the lesions in each of the eight digits were more efficient than methods that did not grade the digits within feet. weighting the ...19957709056
comparative characterization of release factor rf-3 genes of escherichia coli, salmonella typhimurium, and dichelobacter nodosus.the termination of protein synthesis in bacteria requires two codon-specific release factors, rf-1 and rf-2. a gene for a third factor, rf-3, that stimulates the rf-1 and rf-2 activities has been isolated from the gram-negative bacteria escherichia coli and dichelobacter nodosus. in this work, we isolated the rf-3 gene from salmonella typhimurium and compared the three encoded rf-3 proteins by immunoblotting and intergeneric complementation and suppression. a murine polyclonal antibody against e ...19957559341
identification of a gene encoding a bacteriophage-related integrase in a vap region of the dichelobacter nodosus genome.dichelobacter nodosus is the principal causative agent of ovine footrot. nucleotide (nt) sequences from the d. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [katz et al., j. bacteriol. 176 (1994) 2663-2669]. in the present study, the limits of the virulence-associated (va) dna around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to pr ...19957557417
relationship between clinical manifestations of footrot and specific dna products of dichelobacter nodosus amplified through pcr.a total of 141 dichelobacter nodosus isolates from 46 merino sheep farms with various clinical forms of footrot was examined by the gelatin gel test and the polymerase chain reaction (pcr) using virulent (vf2 and vr2) and benign (bf and br) specific primers. isolates from sheep with virulent and high intermediate footrot usually produced relatively thermostable proteases, but a decreasing proportion of the isolates from sheep with medium and low intermediate or benign footrot had thermostable pr ...19958525095
effects of the severity and duration of lesions on the primary and anamnestic humoral responses of sheep to dichelobacter nodosus and observations of natural resistance to footrot.in a flock of 137 sheep naturally infected with dichelobacter nodosus the severity of the lesions was the principal factor associated with the humoral response early in the period of spread of d. nodosus, underrun lesions having the greatest effect. however, after five to six weeks, the duration of underrun lesions rather than their severity or number primarily influenced the response. sheep first affected late in the period of spread had fewer affected feet, milder lesions and a lower humoral r ...19958525101
identification of a native dichelobacter nodosus plasmid and implications for the evolution of the vap regions.studies on the role of various virulence factors of the ovine pathogen, dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of dna into this organism. as an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pjir896, from a clinical isolate. this plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, a198. however, pjir896 lacked the duplicated region present in ...19968654969
further observations on the primary and anamnestic humoral responses to dichelobacter nodosus in sheep in relation to the diagnosis of footrot.an anamnestic serological test for ovine footrot was evaluated. footrot-free lambs were infected with dichelobacter nodosus and treated four, six or eight weeks later. there were strong linear correlations between the severity of the lesions and both the primary response and the anamnestic response evoked by the subcutaneous injection of an antigen from d nodosus 16 weeks after the treatment of the lambs; the latter correlation was stronger than the correlations reported elsewhere in mature shee ...19968685533
assessment of gelatin gel and elastase tests for detection of protease activity of dichelobacter nodosus isolates from ovine footrot.protease tests (the gelatin gel protease thermostability test, the elastase test or both) were performed on 4296 isolates of dichelobacter nodosus derived from 452 outbreaks of ovine footrot occurring in new south wales. both tests showed a high level of repeatability. in the gelatin gel test, culture broths were heated for 16 min at 66.8 degrees c. heated broths containing thermostable protease digested gelatin (positive gelatin gel test) while those broths containing thermolabile protease fail ...19968870192
comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of dichelobacter nodosus.in a collaborative study that involved four australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pjir318, pjir314b, and pjir313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. a total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. the results showed that 87% of the virulent isolates belo ...19968914257
functional expression of heterologous type 4 fimbriae in pseudomonas aeruginosa.type 4 fimbriae are surface organelles produced by a wide range of bacterial pathogens. in pseudomonas aeruginosa they are associated with a form of surface translocation known as twitching motility and have also been implicated as the receptor for a number of fimbrial-specific bacteriophages. the infrastructural machinery required for type 4 fimbrial biogenesis appears to be conserved as heterologous subunits from other species can be expressed in p. aeruginosa. all of these studies have, until ...19968917091
manipulation of the helper t cell response to influence antigenic competition occurring with a multivalent vaccine.the reduction in antibody observed following inoculation with multiple heterologous dichelobacter nodosus pili antigens is thought to be due to competition between antigen-specific b cells for a limited amount of t cell help. we demonstrate here that this competition is not further influenced by the expansion of cross-reactive antibody secreting cells at the expense of serogroup specific antibody secreting cells. the t cell determinants of pili recognized by sheep and balb/c mice have been defin ...19968934658
organization of ribosomal rna genes from the footrot pathogen dichelobacter nodosus.southern hybridization analysis revealed that there were three rrn loci within the genome of dichelobacter nodosus, the causative organism of ovine footrot. these loci (rrna, rrnb and rrnc) were isolated on recombinant lambda clones, and comprised 16s, 23s and 5s rrna genes closely linked in that order. sequence and primer extension analysis revealed the presence of putative genes encoding trna(ile) and trna(ala) within the 16s-23s spacer region, as well as a number of potential regulatory featu ...19968936315
immunoblot and ultrastructural analysis of antigens extracted from dichelobacter nodosus with potassium thiocyanate.the antigens extracted from dichelobacter nodosus with potassium thiocyanate were analyzed by western blotting with sera from footrot-free sheep and from sheep infected with d. nodosus to identify components specific for infection. several components with molecular mass < 33 kd were associated with infection, particularly bands of 32.5 kd, 30.5 kd, and 28 kd. components with molecular mass > 56 kd may be responsible for false-positive reactions observed when sera of footrot-free sheep react with ...19968844574
the molecular genetics of type-4 fimbriae in pseudomonas aeruginosa--a review.type-4 fimbriae (or pili) are filaments found at the poles of a wide range of bacterial pathogens, including neisseria gonorrhoeae, moraxella bovis, dichelobacter nodosus and pseudomonas aeruginosa. they are composed of a small subunit which is highly conserved among different species and appear to mediate adhesion and translocation across epithelial surfaces via a phenomenon termed "twitching motility'. these fimbriae are key host colonisation factors and important protective antigens. we have ...19968955641
virulence regions and virulence factors of the ovine footrot pathogen, dichelobacter nodosus.ovine footrot is a debilitating and highly infectious disease that is primarily caused by the gram-negative, anaerobic bacterium dichelobacter nodosus. the major antigens implicated in virulence are the type iv fimbriae and extracellular proteases. the fimbriae show sequence and structural similarity to other type iv fimbriae, this similarity extends to genes that are involved in fimbrial biogenesis. several acidic and basic extracellular serine proteases are produced by both virulent and benign ...19968961550
effect of adjuvants on antibody responses of sheep immunised with recombinant pili from dichelobacter nodosus.to compare the effects of two oil emulsion adjuvants (incomplete freunds adjuvant and a proprietary oil adjuvant), deae-dextran, l-tyrosine particles and quil a on the humoral immune responses of sheep immunised with recombinant pili of dichelobacter nodosus (strain a).19969006863
antigens for serological diagnosis of ovine footrot.an antigen extracted from dichelobacter nodosus with potassium thiocyanate (kscn) is currently used in enzyme-linked immunosorbent assay (elisa) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. other antigens were therefore evaluated for use in this test. structural components of the cell envelope of d. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured d. nodosus ...19979100327
improved laboratory diagnosis of ovine footrot: an update.ovine footrot is a complex clinical disease syndrome primarily resulting from infection by the anaerobic bacterium dichelobacter nodosus. in order to aid clinical diagnosis, various laboratory tests based on the detection and measurements of phenotypic properties of d. nodosus have been developed for genus-specific detection as well as virulence determination. however, these tests are generally time-consuming, and tend to be variable with external factors that affect the growth and metabolism of ...19979125362
high-level genetic diversity in the vapd chromosomal region of helicobacter pylori.helicobacter pylori isolates from different patients are characterized by diversity in the nucleotide sequences of individual genes, variation in genome size, and variation in gene order. genetic diversity is particularly striking in vacuolating cytotoxin (vaca) alleles. in this study, five open reading frames (orfs) were identified within a 4.2-kb region downstream from vaca in h. pylori 60190. one of these orfs was closely related to the virulence-associated protein d (vapd) gene of dichelobac ...19979139899
physical and genetic map of the chromosome of dichelobacter nodosus strain a198.a physical map of the chromosome of dichelobacter nodosus strain a198 was constructed using the restriction endonucleases eagi and stui. mapping data indicated the presence of a single, circular chromosome of 1.54 mb. the three rrna operons and the virulence related locus (vrl) were precisely positioned at the junctions of eagi and stui fragments, and their transcriptional orientations were also determined. other d. nodosus genes were assigned to specific eagi and stui fragments. analysis of the ...19979031642
analysis of sequences flanking the vap regions of dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element.dichelobacter nodosus is the causative agent of ovine footrot. the vap regions of the d. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. the virulent d. nodosus strain a198 has multiple copies of the vap regions. in the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain a198 were analysed by southern blotting and dna sequencing. the results suggest that vap regions 1 and 2 rose by independent integration eve ...19979043132
identification, purification, and characterization of the type 4 fimbriae of pasteurella multocida.the presence of fimbriae on pasteurella multocida has been reported, but there have been no prior studies aimed at conclusively characterizing these structures. we now report on the identification and characterization of type 4 fimbriae on serogroup a, b, and d strains of p. multocida. under microaerophilic conditions p. multocida showed an increased expression of the fimbriae, which were observed to form bundles. fimbriae purified by high-performance reverse-phase liquid chromatography constitu ...19978975936
post-translational modifications of meningococcal pili. identification of common substituents: glycans and alpha-glycerophosphate--a review.neisseria meningitidis elaborate filamentous adhesins, pili or fimbriae, which belong to the type-4 structural group of pili also found on other bacterial pathogens such as neisseria gonorrhoeae, pseudomonas aeruginosa, moraxella bovis and dichelobacter nodosus. meningococcal pili readily undergo structural variations which arise as a result of inter- and intra-genomic recombinational events. structural variations often result in variations in bacterial adhesion mediated by pili. studies on stru ...19979224884
ulcerative pododermatitis in free-ranging african elephant (loxodonta africana) in the kruger national park.the occurrence of severe lameness in adult african elephant bulls in a shrub mopane (colophospermum mopane) ecosystem was investigated. large ulcers in the soles of at least one front foot were seen in each of the recorded cases. microscopically, the lesion can be described as a severe, chronic-active, ulcerative, bacterial pododermatitis (complicated by hypersensitivity/septic vasculitis). a variety of bacteria were isolated from these lesions as well as from regional lymph nodes. streptococcus ...19979204500
plasmid maintenance functions of the large virulence plasmid of shigella flexneri.the large virulence plasmid pmysh6000 of shigella flexneri contains a replicon and a plasmid maintenance stability determinant (stb) on adjacent sali fragments. the presence of a repfiia replicon on the sali c fragment was confirmed, and the complete sequence of the adjacent sali o fragment was determined. it shows homology to part of the transfer (tra) operon of the f plasmid. stb stabilizes a partition-defective p1 miniplasmid in escherichia coli. a 1.1-kb region containing a homolog of the f ...19979171415
aetiology of ovine footrot in spain.four hundred and sixty strains of obligate anaerobes were isolated from 216 cases of ovine foot rot distributed throughout spain. the predominant species was dichelobacter nodosus, which was isolated in 168 cases (77.8 per cent). a higher proportion of the strains of d nodosus than of the other strains had elastolytic activity, 118 of the 168 strains degrading elastin. species belonging to the genus prevotella were isolated from 96 cases (44.4 per cent). serotyping of the d nodosus strains showe ...19989481841
complementation analysis of the dichelobacter nodosus fimn, fimo, and fimp genes in pseudomonas aeruginosa and transcriptional analysis of the fimnop gene region.the causative agent of ovine footrot, the gram-negative anaerobe dichelobacter nodosus, produces polar type iv fimbriae, which are the major protective antigens. the d. nodosus genes fimn, fimo, and fimp are homologs of the pseudomonas aeruginosa fimbrial assembly genes, pilb, pilc, and pild, respectively. both the pild and fimp genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. to investigate the functional similarity of ...19989423871
electroporation-mediated transformation of the ovine footrot pathogen dichelobacter nodosus.studies on the potential virulence genes of the ovine footrot pathogen dichelobacter nodosus have been hindered by the lack of a genetic system for this organism. in an attempt to accomplish the transformation of d. nodosus cells, we constructed a plasmid that contained part of a native d. nodosus plasmid and carried a tetracycline resistance gene that was located between the d. nodosus rrna promoter and terminator. this plasmid was used to transform several d. nodosus strains to tetracycline re ...19989868785
purification and characterization of a putative fimbrial protein/receptor of porphyromonas gingivalis.porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, p. gingivalis is a gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein. virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes. a 43 kda fimbrillin from p. gingivalis has been isolated and characterized. however, there is evid ...19989612983
molecular analysis of dichelobacter nodosus isolated from footrot in sheep in malaysia.pulsed field gel electrophoresis analysis of genomic dna was used to investigate genetic diversity among dichelobacter nodosus from footrot in sheep in malaysia. twelve dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as dichelobacter nodosus by polymerase chain reaction technique using the species-specific dichelobacter nodosus 16s rna sequence ac and c as primers. pulsed field gel electrophoresis banding profiles using restriction enzymes apai (5' ...19989791871
isolation of spirochaetes from an incident of severe virulent ovine footrot. 19989921625
severity and persistence of footrot in merino sheep experimentally infected with a protease thermostable strain of dichelobacter nodosus at five sites.to test the hypothesis that ovine footrot associated with a thermostable protease strain of dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment.19989578765
the gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in staphylococcus aureus.tst, the gene for toxic shock syndrome toxin-1 (tsst-1), is part of a 15.2 kb genetic element in staphylococcus aureus that is absent in tsst-1-negative strains. the prototype, in rn4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a dichelobacter nodosus vape homologue and a putative integrase. it is readily transferred to a reca recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same ...19989720870
serological classification and virulence determination of dichelobacter nodosus isolated from alberta and british columbia sheep.ovine footrot is a contagious disease of sheep that occurs in temperature climates. it is caused by the strict anaerobe, dichelobacter nodosus. benign and virulent organisms are differentiated according to serotype and protease production. this study was conducted to identify the presence of virulent serotypes of d. nodosus in sheep flocks in alberta and british columbia. dichelobacter nodosus was detected in lame sheep from 11 of 15 (73%) flocks in alberta and in 4 of 5 (80%) british columbia f ...19989442937
biodegradable implants for the delivery of veterinary vaccines: design, manufacture and antibody responses in sheep.biodegradable implants made from cholesterol and lecithin (c:l) were used to deliver a recombinant antigen (recombinant dichelobacter nodosus pili) and adjuvant (quil a) to sheep. implants (5.5- x 1.8-mm) were placed subcutaneously and compared to a conventional vaccination regime (2 injections, 4 weeks apart) for antibody responses and tissue compatibility. release profiles of antigen and adjuvant were also studied in vitro and in vivo. the presence of quil a in vaccine implants had a marked ef ...19989685925
identification and characterisation of serogroup m among nepalese isolates of dichelobacter nodosus, the transmitting agent of footrot in small ruminants.one thousand and sixty three isolates of dichelobacter nodosus cultured between 1992 and 1996 from cases of footrot in sheep and goats of migratory flocks of nepal were characterised by agglutination test using prototype antisera of the australian classification system. of those, sixty six isolates could not be classified into any of the nine serogroups (a-i). this study was therefore undertaken to characterise these isolates. it was established that they were agglutinated by antiserum against s ...19989791869
effectiveness of small workshops for improving farmers' knowledge about ovine footrot.to determine sheep farmers' attitudes to and beliefs about ovine footrot, and to improve their knowledge about the diagnosis, control and eradication of this disease.199910376104
genetic characterisation of protective vaccine responses in sheep using multi-valent dichelobacter nodosus vaccines.protective vaccine responses to nine distinct serogroups of dichelobacter nodosus (serogroups a-i) can be readily measured by serogroup-specific k-agglutinating antibody titres. on the basis of a large quantitative genetic experiment (1200 progeny from 129 sire groups), it was shown that variation in antibody responses following vaccination with a multi-valent pilus antigen d. nodosus vaccine (serogroups a-i) is, in part, under genetic control and thus heritable. based on the genetic relationshi ...199910614512
severe ovine foot disease. 199910619613
transmission of virulent footrot between sheep and goats.to determine the infectivity of ovine and caprine strains of dichelobacter nodosus for both sheep and goats.199910451731
pcr-rflp of outer membrane proteins gene of dichelobacter nodosus: a new tool in the epidemiology of footrot.currently only phenotypic epidemiological markers, serogrouping and virulence testing of dichelobacter nodosus, are available for investigating footrot outbreaks in small ruminants. these methods have limitations in tracing the source of infection. in this study, a genotypic marker, pcr-rflp of outer membrane protein gene, was used to characterize d. nodosus. the technique was evaluated in a controlled experiment involving two strains of bacteria. pcr-rflp was found to be highly specific in diff ...199910459657
the site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in dichelobacter nodosus, the causative agent of ovine footrot.the gram-negative anaerobe dichelobacter nodosus is the causative agent of footrot in sheep. the authors have previously characterized two genetic elements, the inta (vap) and intb elements, which integrate into the genome of d. nodosus. in the virulent strain a198 there are two copies of the inta element. one copy is integrated into the 3' end of the trna-sergcu gene, close to the aspartokinase (aska) gene, and the second copy is integrated into the 3' end of the trna-sergga gene, next to the p ...199910537206
eikenella corrodens phase variation involves a posttranslational event in pilus formation.the human pathogen eikenella corrodens synthesizes type iv pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. on solid medium, piliated variants form small (s-phase), corroding colonies whereas nonpiliated variants form large (l-phase), noncorroding colonies. we are studying the molecular basis of this phase variation in the clinical isolate e. corrodens va1. a genomic fragment encoding the major type iv pilin was cloned from the s-ph ...199910400570
identification of a virulence-associated protein homolog gene and isra1 in a plasmid of riemerella anatipestifer.riemerella anatipestifer is the causative agent of polyserositis of ducks and geese. we have previously reported that a 3.9-kb plasmid, pcfc1, carries protein genes (vapd1 and vapd2) that are similar to virulence-associated genes of other bacteria. in the present study, we report the complete sequence of a second plasmid of 5.6 kb, pcfc2. pcfc2 has a 28% g-c content and three large open reading frames (orfs). one of the orfs (designated asvapd1) encodes a polypeptide that shares 53.9, 53.9, 48.3 ...199910481080
update on footrot in south-west germany.82 dichelobacter nodosus strains isolated from 9 footrot affected sheep flocks in south west germany were serotyped and tested for virulence. serovar b was present in all flocks, representing 64.4% of all isolated d. nodosus field strains. other serovares found were type a, c, e, g and h. virulent strains were identified in 5 flocks, while intermediate strains were isolated from all 9 flocks. all serological untypeable strains proved to be avirulent. based on these epidemiological findings the u ...199910085580
a bacteriophage encoding a pathogenicity island, a type-iv pilus and a phage receptor in cholera bacteria.the virulence properties of many pathogenic bacteria are due to proteins encoded by large gene clusters called pathogenicity islands, which are found in a variety of human pathogens including escherichia coli, salmonella, shigella, yersinia, helicobacter pylori, vibrio cholerae, and animal and plant pathogens such as dichelobacter nodosus and pseudomonas syringae. although the presence of pathogenicity islands is a prerequisite for many bacterial diseases, little is known about their origins or ...199910360577
complete nucleotide sequence of the 27-kilobase virulence related locus (vrl) of dichelobacter nodosus: evidence for extrachromosomal origin.the vrl locus is preferentially associated with virulent isolates of the ovine footrot pathogen, dichelobacter nodosus. the complete nucleotide sequence of this 27.1-kb region has now been determined. the data reveal that the locus has a g+c content much higher than the rest of the d. nodosus chromosome and contains 22 open reading frames (orfs) encoding products including a putative adenine-specific methylase, two potential deah atp-dependent helicases, and two products with sequence similarity ...199910024571
identification and grouping of dichelobacter nodosus, using pcr and sequence analysis.dichelobacter nodosus is the causative agent of ovine foot rot, a disease that is a constant economic burden for many western sheep ranches. vaccination is one method of treating foot rot. a higher and more specific immune response is observed when monovalent vaccines are used to treat foot rot, as compared to multivalent vaccines, which incorporate all 10 major new zealand d. nodosus serogroups. there is no single assay for specifically identifying and grouping d. nodosus for the purpose of inc ...199910024434
identification and characterization of a native dichelobacter nodosus plasmid, pdn1.the gram-negative anaerobe dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. we report here the characterization of a novel native plasmid, pdn1, from d. nodosus. sequence analysis has revealed that pdn1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to escherichia coli incompatibility group q. however, in contrast to these plasmids, pdn1 encodes no antibiotic resistance determinants, lacks genes e ...200010783302
extensive diversity in new zealand dichelobacter nodosus strains from infected sheep and goats.footrot is a contagious bacterial disease of ruminants spread by the gram-negative, anaerobic organism, dichelobacter nodosus. it is endemic in new zealand and throughout sheep and goat farming regions of the world. using the polymerase chain reaction (pcr) to amplify fragments of the fimbrial gene (fima), d. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within new zealand. dna sequencing revealed 15 strains covering eight serogroups on the new zealand farms. the pr ...200010665539
type iv pilus genes pila and pilc of pseudomonas stutzeri are required for natural genetic transformation, and pila can be replaced by corresponding genes from nontransformable species.pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. after transposon mutagenesis, transformation-deficient mutants were isolated from a p. stutzeri jm300 strain. in one of them a gene which coded for a protein with 75% amino acid sequence identity to pilc of pseudomonas aeruginosa, an accessory protein for type iv pilus biogenesis, was inactivated. the presence of type iv pili was demonstrated by susceptibility to the type iv pilus-dep ...200010735861
occurrence of different strains of dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections.to investigate the occurrence of s1, u1 and t strains of dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections.200010840576
novel fimbrial subunit genes of dichelobacter nodosus: recombination in vivo or in vitro?polymerase chain reaction (pcr) was used to amplify the variable region of the fimbrial subunit encoding gene (fima) of dichelobacter nodosus from sheep and goats infected with footrot. two amplimers (designated x and y) generated single-strand conformation polymorphism (sscp) patterns different to those of previously identified serogroups and serotypes. dna sequencing revealed that these two fragments were novel. the upstream of x (nt 1-183) was identical to serotype m1 while its downstream (nt ...200010946146
3'overhangs influence pcr-sscp patterns. 200011084854
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