Publications

TitleAbstractYear
Filter
PMID
Filter
molecular detection and characterization of dichelobacter nodosus in ovine footrot in india.dichelobacter nodosus was detected in three clinical cases of ovine footrot in kashmir, india. the detection was done by pcr in three clinical specimens directly, without isolating the organism, using species-specific 16s rdna primers. positive results were indicated by amplification of a 783 bp product. all the three samples were subjected to serogrouping by multiplex pcr using group (a-i) specific primers. all the three samples revealed the presence of serogroup b of d. nodosus by yielding a s ...200415294315
comparative study of biochemical methods for the identification of dichelobacter nodosus.the aim of this study was to compare four identification procedures to detect dichelobacter nodosus and develop a rapid, simple and effective method to identify d. nodosus strains isolated from cases of ovine footrot. the four methods used were: (a) the classic guidelines set down by holdeman et al. (1977) and summanen et al. (1993) which are based on gas liquid chromatography (glc) and different biochemical tests, this method was considered as landmark; (b) baron and citron's flowchart for the ...200415379442
effectiveness of different adjuvants in stimulating dichelobacter nodosus antibody in sheep vaccinated against ovine footrot.this research consists of an evaluation of the effectiveness of different substances administered as adjuvants in the stimulation of humoral immune response induced by the vaccine composed of strains a1, a2 and c of dichelobacter nodosus. to do this, a total of 120 merino sheep were vaccinated and revaccinated. these sheep were selected from a farm located in the region of extremadura (spain), and they were divided into 12 groups of 10 animals each. an additional group with 10 sheep was used as ...200415379443
identification of a dichelobacter nodosus ferric uptake regulator and determination of its regulatory targets.the expression of iron regulated genes in bacteria is typically controlled by the ferric uptake regulator (fur) protein, a global transcriptional repressor that regulates functions as diverse as iron acquisition, oxidative stress, and virulence. we have identified a fur homologue in dichelobacter nodosus, the causative agent of ovine footrot, and shown that it complements an escherichia coli fur mutant. homology modeling of the d. nodosus fur protein with the recently solved crystal structure of ...200515601721
comparative study of three surgical treatments for two forms of the clinical presentation of bovine pododermatitis.in this study, 1013 animals showing signs of clinical pododermatitis were examined and divided into five unevenly numbered groups. affected animals in groups i and ii showed only signs of vegetative interdigital pododermatitis. the lesions were surgically removed and either protected with bandages (in group i) or cauterized with incandescent iron (group ii). the animals in groups iii, iv, and v, showed signs of necrotic pododermatitis. these were treated with different protocols after the necrot ...200415604479
current approaches to the management of ovine footrot.ovine footrot remains the most important cause of lameness in sheep flocks in the uk, despite the existence of proven methods for the control of the disease. recent research suggests that sheep farmers may be unaware of these methods and may allocate greater resources to treatment of footrot rather than to its prevention. foot paring, topical treatments, vaccination and parenteral antibiotic therapy all have a role in treating sheep with advanced footrot infections, but prevention of severe infe ...200515683762
plasmid-borne florfenicol resistance in pasteurella multocida.a florfenicol-resistant pasteurella multocida isolate from a calf was investigated for the genetic basis of florfenicol resistance and the location of the resistance gene.200515814600
the detection and characterisation of dichelobacter nodosus from cases of ovine footrot in england and wales.footrot, caused by the strictly anaerobic bacterium dichelobacter nodosus, is the most common cause of lameness in sheep in great britain but problems exist in association with its diagnosis and control. the fastidious nature of d. nodosus means that complex media and several weeks are required for characterisation. an alternative method to simplify and enhance the detection of d. nodosus in clinical samples is therefore highly desirable. in terms of control, anecdotal evidence from the farming ...200515917134
the use of melatonin as a vaccine agent.molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. in seasonally-breeding animals, the indolamine is able to improve reproductive performance. with the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in ...200516055232
update on ovine footrot in new zealand: isolation, identification, and characterization of dichelobacter nodosus strains [corrected].dichelobacter nodosus, a gram-negative strict anaerobe, is the essential causative agent of ovine footrot. despite its worldwide presence, the disease has significant economic impact in those sheep-farming countries with a temperate climate and moderate to high rainfall, such as new zealand (nz) and australia. in this study, we aimed to isolate, identify, and characterize as many d. nodosus strains as possible from nz farms by using polymerase chain reaction (pcr)-based technology. understanding ...200516280202
the occurrence of treponemes in contagious ovine digital dermatitis and the characterisation of associated dichelobacter nodosus.contagious ovine digital dermatitis (codd) is a recently recorded, apparently new infection of the ovine hoof, which differs clinically from footrot caused by dichelobacter nodosus and which fails to respond well to accepted treatment practices for footrot. despite the welfare implications of such an infection, very little research has been performed on codd to date and the aetiology remains confused. suggestions have been made that there is a potential role for treponemes in the pathogenesis of ...200516280206
recent footrot outbreak in debrezeit swine farm, central ethiopia.an outbreak of footrot has occurred in debrezeit swine farm that is located 44 km south east of the capital. among 24 pigs, 75% showed lameness, hot, painful and swollen feet, hemorrhagic bruising of the coronary band, heel erosion, sole ulcers and separation of skin from hoof on hind limbs. bacteriological examination of the specimen from the affected limbs has shown the involvement of staphylococcus, streptococcus and dichelobacter nodosus. the overt clinical lameness and inability to stand or ...200516294005
eradication of virulent footrot from sheep and goats in an endemic area of nepal and an evaluation of specific vaccination.programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region of nepal. from 1993 to 1996 vaccination against two endemic virulent strains of dichelobacter nodosus was tested for its potential to contribute to the eradication of footrot from the region. only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before the ...200212243270
improved diagnosis of virulent ovine footrot using the inta gene.footrot is a mixed bacterial infection of the hooves of sheep. the gram-negative anaerobic bacterium dichelobacter nodosus is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. in australia, in the state of new south wales (nsw), only virulent footrot is subject to regulatory action, including quarantine. however, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection ...200616716540
regulation of type iv fimbrial biogenesis in dichelobacter nodosus.type iv fimbriae are expressed by several bacterial pathogens and are essential for virulence in dichelobacter nodosus, which causes ovine footrot. we have identified a two-component signal transduction system (pilr/s) and an alternative sigma factor (sigma 54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in d. nodosus. western blots showed that in both pilr and rpon mutants, fimbrial subunit production was significantly reduced by a proces ...200616788189
activity and cellular localization of amylases of rabbit cecal bacteria.five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) cfu/g. the activity and cellular localization of amylases was determined in 9 bacteria identified as actinomyces israeli (strains aa2 and ad4), bacteroides spp. (strain aa3), dichelobacter nodosus (strain aa4), mitsuokella multiacidus (strain aa6), eubacterium spp. (strains aa7 and ab2), clos ...200617007433
aetiology of caprine footrot in extremadura region, spain.the present study reports on the location of major foci of footrot in goats in the extremadura region of spain by the determination of locally occurring strictly anaerobic microorganisms involved in the pathogenesis and development of this disease. the most commonly isolated microorganisms belonged to the genera dichelobacter, fusobacterium, porphyromonas and prevotella; these were found in conjunction with other species of minor importance. the species most frequently isolated were fusobacteriu ...200617020135
isolation and characterization of dichelobacter nodosus from ovine and caprine footrot in kashmir, india.footrot is a highly contagious and economically important disease of sheep and goats, caused by dichelobacter nodosus, a slow growing anaerobic gram-negative rod. the current australian antigenic classification system, based on variation in the fimbriae, classifies d. nodosus into at least 10 serogroups (a-i and m) and 18 serotypes. this investigation was intended to determine the serological diversity of d. nodosus in this region of kashmir, india. exudates of footrot lesions were collected fro ...200717250860
diagnosis of footrot in goats: application of elisa tests for response to antigens of dichelobacter nodosus.goats are an important natural host for footrot and are infected with dichelobacter nodosus that have virulence characteristics similar to those of sheep strains. however, the humoral response of goats to d. nodosus antigens and the possibility of a serological diagnosis of footrot in goats have not been studied. with the aim of evaluating a diagnostic elisa test, we investigated the primary immune response of goats to experimental and natural infection, the memory response in recovered animals, ...200212052334
in vitro antimicrobial susceptibility of anaerobic bacteria isolated from caprine footrot.the in vitro antimicrobial susceptibility of gram-negative anaerobic bacilli commonly isolated from footrot in goats was studied. a total of 97 isolates belonging to the genera dichelobacter, fusobacterium, prevotella, porphyromonas and bacteroides, obtained from clinical cases of footrot in south-western spain between march 2000 and may 2001, were tested against 25 antimicrobial agents comprising beta-lactams, aminoglycosides, macrolides, chloramphenicol, quinolones, lincosamides, sulphonamides ...200717385552
rapid and accurate typing of dichelobacter nodosus using pcr amplification and reverse dot-blot hybridisation.here we describe an approach to genotyping d. nodosus, based on variation in the fimbrial subunit gene (fima), which uses polymerase chain reaction (pcr) amplification and hybridisation to immobilised oligonucleotides (pcr/oligotyping). the variable region of d. nodosus fima, amplified and labelled with digoxigenin (dig) in a single multiplex pcr amplification, was hybridised to a panel of group- and type-specific poly-dt tailed oligonucleotides that were immobilised on a nylon membrane strip. a ...200111295335
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
type iv fimbrial biogenesis is required for protease secretion and natural transformation in dichelobacter nodosus.the objective of this study was to develop an understanding of the molecular mechanisms by which type iv fimbrial biogenesis, natural transformation, and protease secretion are linked in the ovine foot rot pathogen, dichelobacter nodosus. we have shown that like the d. nodosus fimbrial subunit fima, the pilin-like protein pile and the fimn, fimo, and fimp proteins, which are homologs of pilb, pilc, and pild from pseudomonas aeruginosa, are essential for fimbrial biogenesis and natural transforma ...200717513472
glycosylation of type-iv fimbriae of dichelobacter nodosus.dichelobacter nodosus is the causative agent of ovine footrot and the type-iv fimbriae on this bacterium are essential for maintaining its virulence. in this study, we reveal that these fimbriae are glycosylated. this was demonstrated in several ways: by the detection of carbohydrate on fimbrial protein using periodic acid schiff reagent (pas) staining of sds-page gels and by demonstrating enzymatic deglycosylation and by analysis of the amino acid sequences derived from the fima gene, whereby t ...200817681435
analyses of the vrl gene cluster in desulfococcus multivorans: homologous to the virulence-associated locus of the ovine footrot pathogen dichelobacter nodosus strain a198.major parts of the virulence-associated vrl locus known from the gammaproteobacterium dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium desulfococcus multivorans. in the genome of d. multivorans 13 of the 19 vrl genes described for d. nodosus are present and highly conserved with respect to gene sequence and order. the vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of ...200717693723
twitching motility is essential for virulence in dichelobacter nodosus.type iv fimbriae are essential virulence factors of dichelobacter nodosus, the principal causative agent of ovine foot rot. the fima fimbrial subunit gene is required for virulence, but fima mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type iv fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. we showed that mutation of either the pilt or pilu gene eliminated the ability to carry out twitchin ...200818310333
the complete sequence and functional analysis of panl, the large plasmid of the unicellular freshwater cyanobacterium synechococcus elongatus pcc 7942.two endogenous plasmids are present in synechococcus elongatus pcc 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. the large plasmid, panl, was shown previously to be involved in adaptation of s. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. here, we report the complete sequence of panl, which is 46,366 bp in length with 53% gc content and encodes 58 putative orfs. the panl plas ...200818353436
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
pilot trials in australia on eradication of footrot by flock specific vaccination.footrot is a contagious disease of ruminants requiring strains of dichelobacter nodosus that possess virulence factors including proteases and fimbriae. sheep can be immunised against footrot using vaccine-containing fimbriae, either native or recombinant. the fimbriae are responsible for the serological k-agglutination reaction, which has been used to classify field isolates into nine major serogroups. the range of protection conferred by vaccination is largely restricted to the serogroup invol ...200818621488
proteomic profiling of ovine serum by seldi-tof ms: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples.the diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (seldi-tof ms) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. optimum combinations of proteinchip array surfaces, energy absorbing molecules, sample dilutions and instrument settings w ...201018775566
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
isolation of the bacteriophage dinohi from dichelobacter nodosus and its interactions with other integrated genetic elements.the gram-negative anaerobic pathogen dichelobacter nodosus carries several genetic elements that integrate into the chromosome. these include the inta, intb, intc and intd elements, which integrate adjacent to csra and pnpa, two putative global regulators of virulence and the virulence-related locus, vrl, which integrates into ssra. treatment of d. nodosus strains with ultraviolet light resulted in the isolation of dinohi, a member of the siphoviridae and the first bacteriophage to be identified ...200819088904
melatonin enhances the immune response to vaccination against a1 and c strains of dichelobacter nodosus.melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of a1 and c strains of dichelobacter nodosus in sheep. subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. administration of melatonin to vaccinated animals enhanced both the titer of an ...200919166891
effect of parenteral selenium administration to sheep on prevalence and recovery from footrot.insufficient intake of selenium (se) is common in many regions, and can contribute to increased susceptibility to and prolonged recovery from infectious diseases.200919192142
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
dichelobacter nodosus, fusobacterium necrophorum and the epidemiology of footrot.footrot is a debilitating disease of sheep resulting in lameness, production losses and suffering. to study the basic bacteriology of the disease, a survey was initiated across commercial farms and non-commercial research flocks to compare the bacteriology of symptomatic footrot infected sheep with healthy asymptomatic sheep. of the 80 farmers initially contacted, 14 collected hoof swabs and returned the swabs by post. following dna extraction, species-specific pcr was used to identify if dichel ...200919239925
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
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
identification of two new dichelobacter nodosus strains in germany.the multi-strain anaerobe dichelobacter nodosus causes footrot, a contagious disease of the digits of ruminants that occurs worldwide. eleven d. nodosus-like isolates from footrot lesions of sheep in germany were investigated by pcr amplification of the variable region of the d. nodosus fimbrial (fima) gene. in six isolates, fima pcr amplicons of a size suggestive of d. nodosus were generated. single-strand conformational polymorphism (sscp) analysis of the amplicons revealed three banding patte ...201019285440
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
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
comparative genomic analysis of ten streptococcus pneumoniae temperate bacteriophages.streptococcus pneumoniae is an important human pathogen that often carries temperate bacteriophages. as part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. all of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis ...200919502408
discrimination between bacteroides, dichelobacter, fusobacterium, porphyromonas and prevotella isolated from caprine footrot by pcr-rflp - short communication.footrot is widely considered the most severe and most common foot pathology in small ruminants. this study tested the ability of a molecular typing system based on polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) assay of the 16s rrna gene to discriminate between the strict anaerobe genera most commonly isolated from footrot ( bacteroides, dichelobacter, fusobacterium, porphyromonas and prevotella ) in goats in extremadura (spain), with a view to facilitating identif ...200919584033
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
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
deletion of the c-terminus of polynucleotide phosphorylase increases twitching motility, a virulence characteristic of the anaerobic bacterial pathogen dichelobacter nodosus.the gram-negative anaerobe dichelobacter nodosus is the causative agent of footrot in sheep. different strains of d. nodosus cause disease of differing severities, ranging from benign to virulent. virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. we have identified polynucleotide phosphorylase (pnpase) as a putative virulence regulator and have proposed that pnpase expression is modulated by the adjacent integ ...201019895640
[structural and functional specificity of the dichelobacter nodosus pili. synthesis of the protein pilin of d. nodosus in various bacterial recombinant strains].the ovine foot rot is a severe infectious disease of sheep. dichelobacter nodosus is an essential pathogen of this disease. an obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bacterial density and fastidious growth requirements. this causes obstacles to vaccine production and makes it difficult to diagnose the disease. the diagnosis in this case is more expensive. fimbriae (or pili) are one of the major factors of virulence of d. nodosus. their antigenic ...200920050159
understanding the molecular epidemiology of the footrot pathogen dichelobacter nodosus to support control and eradication programs.the gram-negative anaerobe dichelobacter nodosus is the primary etiologic agent of ovine footrot. few studies of the genetic diversity and epidemiology of d. nodosus have been done, despite the economic cost and welfare implications of the disease. this study examined a large collection of australian isolates; 735 isolates from footrot-infected sheep from 247 farms in western australia (wa) were tested by pulsed-field gel electrophoresis (pfge), and a subset of 616 isolates was tested by infrequ ...201020071558
crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen dichelobacter nodosus.dichelobacter nodosus is the principal causative agent of ovine footrot, a disease of significant economic importance to the sheep industry. d. nodosus secretes a number of subtilisin-like serine proteases which mediate tissue damage and presumably contribute to the pathogenesis of footrot. strains causing virulent footrot secrete the proteases aprv2, aprv5 and bprv and strains causing benign footrot secrete the closely related proteases aprb2, aprb5 and bprb. here, the cloning, purification and ...201020208163
diagnostic sampling strategies for virulent ovine footrot: simulating detection of dichelobacter nodosus serogroups for bivalent vaccine formulation.dichelobacter nodosus is a slow-growing anaerobic bacterium that is the causative agent of virulent ovine footrot. vaccination targeted at up to two specific serogroups can eliminate those serogroups from infected flocks, but requires identification of serogroups present in infected flocks. serogroups can be identified using slide agglutination or polymerase chain reaction (pcr) methods. the objectives of this project were to use stochastic simulation modeling to estimate the efficacy of samplin ...201020303191
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
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
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
ovine footrot: new approaches to an old disease.footrot is a bacterial disease that has substantial economic and welfare impacts in sheep and can be difficult to manage. research is focussed on reducing the impact that footrot has on farmers and their flocks and better understanding the aetiology of the disease. key areas of current research include, developing better vaccines, deploying tailored vaccines in a specific and targeted fashion on individual farms, analysing and developing better farm management practices to suit specific sheep fa ...201020926208
the subtilisin-like protease aprv2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. d. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have bee ...201021124876
footrot on a sheep breeding farm in the himalayan state of jammu and kashmir.in the present study ovine footrot was detected clinically on a sheep farm in the himalayan state of jammu and kashmir. dichelobacter nodosus was confirmed by culture and polymerase chain reaction (pcr) using species-specific 16s ribosomal rna primers. when cultured, the organism appeared as flat colourless colonies having a fine granulated structure with irregular margins, and showing characteristic gram-negative rods with swollen ends. detection by pcr from cultured bacteria resulted in amplif ...201021309465
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
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
ovine pedomics: the first study of the ovine foot 16s rrna-based microbiome.we report the first study of the bacterial microbiome of ovine interdigital skin based on 16s rrna by pyrosequencing and conventional cloning with sanger-sequencing. three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. the sheep were classified as having either healthy interdigital skin (h) and interdigital dermatitis (id) or virulent footrot ...201121430786
prevalence of footrot in swedish slaughter lambs.abstract:201121492433
implications of host genetic variation on the risk and prevalence of infectious diseases transmitted through the environment.previous studies have shown that host genetic heterogeneity in the response to infectious challenge can affect the emergence risk and the severity of diseases transmitted through direct contact between individuals. however, there is substantial uncertainty about the degree and direction of influence owing to different definitions of genetic variation, most of which are not in line with the current understanding of the genetic architecture of disease traits. also, the relevance of previous result ...201121527777
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
s1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction.the ovine footrot pathogen, dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. virulent and benign strains of d. nodosus secrete the basic proteases bprv and bprb, respectively, with the catalytic domain of these enzymes having 96% sequence identity. at present, it is not known how sequence variation between these two putative virulence factors influences their respective ...201121990366
Identification of two new serotypes within serogroup B of Dichelobacter nodosus.The present study records the strain-specific molecular typing system for Dichelobacter nodosus (D. nodosus) based on genetic analysis of fimA locus. Based on the study two new serotypes B5 and B6 are reported within the serogroup B. Out of 200 swab samples collected randomly from foot lesions of footrot affected sheep from all the districts of Kashmir, India, 122 (61.0%) detected positive for D. nodosus. Serogroup B was predominantly prevalent in 83.60% of positive samples. Restriction fragment ...201122197966
plasmid-mediated florfenicol resistance in mannheimia haemolytica isolated from cattle.the aim of this study was to analyse a florfenicol-resistant mannheimia haemolytica isolated from a calf to determine the genetic basis of its florfenicol-resistance. the antimicrobial susceptibility and plasmid content of the isolate were determined. a florfenicol resistant plasmid carrying the flor gene was identified by pcr and transformed into escherichia coli jm109 and hb101 strains. the plasmid was then mapped and sequenced completely. the isolate was resistant to chloramphenicol, florfeni ...201122019290
ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential.pilin proteins assemble into type iv pili (t4p), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. however, challenges in crystallizing full-length fiber-forming and membrane protein pilins leave unanswered questions regarding pilin structures, assembly, functions, and vaccine potential. here we report pilin structures of full-length dnfima from the sheep pathogen dichelobacter nodosus and ftpile fr ...201122027840
impact of footrot vaccination and antibiotic therapy on footrot and contagious ovine digital dermatitis.footrot and contagious ovine digital dermatitis (codd) are common causes of foot disease of sheep in the uk. the study reported here is a split flock randomised treatment trial undertaken on a group of 748 fattening lambs on a uk sheep farm affected by codd and footrot. the sheep were randomly assigned to one of two treatment protocols. in protocol a, all sheep were given two doses of footrot vaccine (footvax, msd), plus targeted antibiotic therapy (long-acting amoxicillin, betamox la, norbrook ...201222266683
high-level association of bovine digital dermatitis treponema spp. with contagious ovine digital dermatitis lesions and presence of fusobacterium necrophorum and dichelobacter nodosus.contagious ovine digital dermatitis (codd) is an important foot disease in sheep, with significant animal welfare and economic implications. it is thought that codd emerged from bovine digital dermatitis (bdd) via treponemal bacteria. with wildlife species such as elk now suffering a codd-like disease, it is imperative to clarify these disease etiologies. a large investigation into treponemal association with codd is warranted. codd lesions (n = 58) and healthy sheep foot tissues (n = 56) were a ...201525740778
development and comparison of a real-time pcr assay for detection of dichelobacter nodosus with culturing and conventional pcr: harmonisation between three laboratories.ovine footrot is a contagious disease with worldwide occurrence in sheep. the main causative agent is the fastidious bacterium dichelobacter nodosus. in scandinavia, footrot was first diagnosed in sweden in 2004 and later also in norway and denmark. clinical examination of sheep feet is fundamental to diagnosis of footrot, but d. nodosus should also be detected to confirm the diagnosis. pcr-based detection using conventional pcr has been used at our institutes, but the method was laborious and t ...201222293440
drug target identification and prioritization for treatment of ovine foot rot: an in silico approach.ovine foot rot is an infection of the feet of sheep, mainly caused by dichelobacter nodosus. in its virulent form, it is highly contagious and debilitating, causing significant losses in the form of decline in wool growth and quality and poor fertility. current methods of treatment are ineffective in complete eradication. effective antibiotic treatment of foot rot is hence necessary to ensure better outcomes during control phases by reduction in culling count and the possibility of carriers of t ...201627379247
secretion of tcpf by the vibrio cholerae toxin-coregulated pilus biogenesis apparatus requires an n-terminal determinant.type iv pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. we and others have shown that type iv pili are important for protein secretion across the outer membrane, similar to type ii secretion systems. this study explored the relationship between protein secretion and pilus formation in vibrio cholerae. the toxin-coregulated pilus (tcp), a type iv pilus required for v. cholerae pathogenesis, is necessary for the secretion of the colonization fac ...201323564177
current understanding of the aetiology and laboratory diagnosis of footrot.footrot is a highly contagious disease of the feet of ruminants caused by the synergistic action of certain bacterial species of which dichelobacter nodosus (d. nodosus) is the main transmitting agent. the infection is specific to sheep and goats, although it has also been reported in cattle, horses, pigs, deer and mouflon. the antigenic diversity of d. nodosus is due to variations in the dna sequence of its fimbrial subunit gene (fima) and provides the basis for classification of the organism i ...200616624707
first study of pathogen load and localisation of ovine footrot using fluorescence in situ hybridisation (fish).analysis of bacterial populations in situ provides insights into pathogen population dynamics and potential reservoirs for disease. here we report a culture-independent study of ovine footrot (fr); a debilitating bacterial disease that has significant economic impact on sheep farming worldwide. disease begins as an interdigital dermatitis (id), which may then progress to separation of the hoof horn from the underlying epidermis causing severe footrot (sfr). dichelobacter nodosus is the causative ...201525742734
reducing exposure to pathogens in the horse: a preliminary study into the survival of bacteria on a range of equine bedding types.to compare the rate of growth of four microbial strains that cause disease in the horse, on four commonly used types of bedding. the moisture-holding capacity of each bedding type was also tested.201727659898
distribution and prevalence of footrot in bhutan.the first cases of footrot in bhutan were reported in sheep in 1990 at the national sheep breeding centre (nsbc), which supplies breeding animals to village sheep flocks throughout bhutan. despite the presence of footrot at the centre the distribution of apparently disease-free sheep continued. cases of footrot were reported in village flocks soon after the disease was diagnosed at nsbc. a national survey was designed to establish the distribution and prevalence of footrot in bhutan. this detect ...200616490719
the use of an autogenous dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in bhutan.an outbreak of virulent footrot was investigated in a flock of 605 merino cross-bred sheep in bhutan. conventional control methods in the preceding eight years had reduced its prevalence from 36-79% in different components of the flock to about 15% overall. only one serogroup (b) of dichelobacter nodosus was identified among 40 isolates cultured from affected sheep. a vaccine prepared from this strain was used in a pilot trial to compare the response of 14 treated and 14 untreated sheep. all aff ...200616019243
minimum inhibitory concentrations for 25 selected antimicrobial agents against dichelobacter nodosus and fusobacterium strains isolated from footrot in sheep of portugal and spain.the agar dilution method was used to determine the inhibitory activity of 25 antimicrobial agents against 69 strains of dichelobacter nodosus and 108 strains of the genus fusobacterium, all of which were isolated from 90 clinical cases of ovine footrot between october 1998 and november 2000. in the case of the micro-organisms belonging to the genus fusobacterium, the six beta-lactams studied (benzyl penicillin, ampicillin, cloxacillin, cefadroxil, cefuroxime and cephalexine) proved to be, in gen ...200415330985
transformation-mediated serogroup conversion of dichelobacter nodosus.dichelobacter nodosus is the essential causative agent of footrot in sheep. the type iv fimbriae of d. nodosus are required for virulence, are highly immunogenic and immunoprotective, and can be divided into 10 major serogroups. fimbrial variation has been postulated to have arisen because of genetic recombination within the fimbrial gene region perhaps as a means of evading the immune response invoked by infection. to show that antigenic variation in these fimbriae could occur after natural tra ...200312488080
detection of dichelobacter nodosus in wild ungulates (capra ibex ibex and ovis aries musimon) and domestic sheep suffering from foot rot using a two-step polymerase chain reaction.severe keratinous hoof afflictions have been recorded in ibex (capra ibex ibex) since 1995 and more recently in mouflon (ovis aries musimon) in switzerland. based on clinical observations and comparison with diseases known to affect domestic ungulates, it was hypothesized these wild ungulates were affected by foot rot associated with infection with dichelobacter nodosus. dichelobacter nodosus has been shown to be the essential pathogen for initiation and establishment of foot rot, a highly conta ...200717347396
assessment of current knowledge of footrot in sheep with particular reference to dichelobacter nodosus and implications for elimination or control strategies for sheep in great britain.a brief summary of the currently accepted aetiology and pathogenesis of footrot and interdigital dermatitis in sheep in great britain is presented together with a description of the transmission dynamics of dichelobacter nodosus. approaches that may be successful for control or elimination of the disease are proposed and areas of uncertainty in current knowledge highlighted. research and practice in great britain is compared with that from other countries and the proposal made that the environme ...200817418598
genome sequence and identification of candidate vaccine antigens from the animal pathogen dichelobacter nodosus.dichelobacter nodosus causes ovine footrot, a disease that leads to severe economic losses in the wool and meat industries. we sequenced its 1.4-mb genome, the smallest known genome of an anaerobe. it differs markedly from small genomes of intracellular bacteria, retaining greater biosynthetic capabilities and lacking any evidence of extensive ongoing genome reduction. comparative genomic microarray studies and bioinformatic analysis suggested that, despite its small size, almost 20% of the geno ...200717468768
cloning, expression, and functional characterization of serine protease aprv2 from virulent isolate dichelobacter nodosus of indian origin.a gene encoding an extracellular protease from dichelobacter nodosus was characterized and expressed in e. coli rosetta-gami (de3). the nucleotide sequence analysis revealed an orf of 1427 bp ecoding 475 amino acids long protein of calculated molecular weight 50.6 kda and pi value 6.09. the phylogenetic analysis showed relatedness to subtilisin-like serine proteases of peptidase s8 family. the amino acid sequence analysis showed presence of n-terminal pre-peptide (1-23 aa), pro-peptide (24-160 a ...201627168406
phylogenetic analysis of dichelobacter nodosus serogroup-specific fima gene from ovine footrot in andhra pradesh.identification of different serogroups of dichelobacter nodosus prevailing in the region and to understand the degree of genetic heterogeneities among the different isolates of d. nodosus.201527047136
infectious lameness among migratory sheep and goats in north-west india, with particular focus on anaerobes.various bacterial species, particularly dichelobacter nodosus, fusobacterium necrophorum, trueperella pyogenes (previously arcanobacterium pyogenes) and treponema spp., have been implicated in infectious conditions manifesting in lameness in sheep and goat populations. the current study reports the causes of infectious lameness in the north-western himalayan region, particularly himachal pradesh (hp), where no such study has been conducted in the past. among a total surveyed population of 27,586 ...201527044157
breeding for resistance to footrot--the use of hoof lesion scoring to quantify footrot in sheep.so that genetic studies can be undertaken on footrot in sheep, it is necessary that a reliable and repeatable method to categorise the phenotype is available. this paper summarises the methods used and results obtained from 1600 hoof lesion scores of 100 mixed-age ewes independently scored twice by two trained operators. using a 5-pont scale describing the severity of foot lesions, residual correlations were used to assess agreement between scorers and scoring occasions. data were analysed using ...200818478350
variation in fusobacterium necrophorum strains present on the hooves of footrot infected sheep, goats and cattle.footrot is a disease of sheep, goats and cattle that causes losses in production and raises welfare issues world-wide. the disease is characterised by destruction of the hard keratin of the hoof leading to lameness, and both dichelobacter nodosus (d. nodosus) and fusobacterium necrophorum (f. necrophorum) are thought to be involved in the etiology of this disease. while a lot is known about the genetic diversity of d. nodosus, very little is known about variation in f. necrophorum, especially as ...200919019570
melatonin, as an adjuvant-like agent, enhances platelet responsiveness.melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. the present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against dichelobacter nodosus (a1 and c serotypes), the bacterium that ca ...200919196436
the detection of dichelobacter nodosus and fusobacterium necrophorum from footrot lesions in new zealand goats. 200919248838
draft genome sequence of dichelobacter nodosus atcc 25549, strain vpi 2340 [11342], a bacterium causing footrot in sheep.we report a draft genome sequence for dichelobacter nodosus atcc 25549, strain vpi 2340 [11342], a causative agent of ovine footrot. the draft genome shares ~98% gene similarity with the available genome of d. nodosus strain vcs1703a but is differentiated by extensive gene duplication and the absence of 13 particular genes.201526404593
the detection of dichelobacter nodosus and fusobacterium necrophorum from ovine footrot in kashmir, india.in a study conducted, a total of 450 swab samples from footrot lesions of naturally infected sheep were collected in all the ten districts of the kashmir valley and were examined for the presence of dichelobacter nodosus (d. nodosus) and fusobacterium necrophorum (f. necrophorum), in order to determine if f. necrophorum was associated with ovine footrot. the detection of f. necrophorum and d. nodosus was carried out by polymerase chain reaction targeting the leukotoxin (lkta) and 16s rrna genes, ...201526159406
association of treponeme species with atypical foot lesions in goats.five uk goat farms with high levels of lameness (prevalence 14-67 per cent) were investigated. on two farms (farms 1 and 2), the animals presented with typical footrot lesions. the remaining three farms (farms 3, 4 and 5) presented with infected lesions on the foot that did not resemble footrot. these lesions were observed to start from the white line or sole but the interdigital space was rarely affected. swabs were processed by pcr to assess the presence of dichelobacter nodosus and three spec ...201525977404
the role of the environment in transmission of dichelobacter nodosus between ewes and their lambs.dichelobacter nodosus (d. nodosus) is the essential causative agent of footrot in sheep. the current study investigated when d. nodosus was detectable on newborn lambs and possible routes of transmission. specific qpcr was used to detect and quantify the load of d. nodosus in foot swabs of lambs at birth and 5-13 h post-partum, and their mothers 5-13 h post-partum; and in samples of bedding, pasture, soil and faeces. d. nodosus was not detected on the feet of newborn lambs swabbed at birth, but ...201525953734
a molecular epidemiology of treponemes in beef cattle digital dermatitis lesions and comparative analyses with sheep contagious ovine digital dermatitis and dairy cattle digital dermatitis lesions.bovine digital dermatitis (bdd) is an infective foot disease commonly reported in dairy cattle where treponema are considered as the primary causative infectious agents. there still remains little definitive information on the etiology of bdd in beef cattle suggesting further investigations are warranted. beef bdd lesions (n=34) and healthy beef foot tissues (n=38) were analysed by pcr for three bdd-associated treponema phylogroups and also for dichelobacter nodosus and fusobacterium necrophorum ...201525937315
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
serological diversity and virulence determination of dichelobacter nodosus from footrot in india.one hundred and twenty-eight swab samples from footrot lesions of naturally infected sheep were examined for presence of dichelobacter nodosus (d. nodosus). the detection of d. nodosus was carried out by polymerase chain reaction (pcr), directly from swabs or after isolation, using 16s rdna specific primers. the isolation of the bacterium was carried out anaerobically on trypticase-arginine-serine (tas) agar containing 4% hoof powder. serogrouping of the d. nodosus was accomplished with multiple ...200919272343
the intd mobile genetic element from dichelobacter nodosus, the causative agent of ovine footrot, is associated with the benign phenotype.the gram-negative anaerobic pathogen dichelobacter nodosus is the principal causative agent of footrot in sheep. the inta, intb and intc elements are mobile genetic elements which integrate into two trna genes downstream from csra (formerly glpa) and pnpa in the d. nodosus chromosome. csra homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of pnpa. we have proposed a model in which virulence in d. nodosus is controll ...200919327404
detection of fusobacterium necrophorum and dichelobacter nodosus in lame cattle on dairy farms in new zealand.lameness in the dairy industry in new zealand causes a problem in lost production, animal welfare and associated costs. to understand what bacteria may be present on the hooves of lame dairy cattle in this grass-fed system, samples were scraped from lame dairy cows and examined for the presence of fusobacterium necrophorum (f. necrophorum) and dichelobacter nodosus (d. nodosus) using the polymerase chain reaction (pcr). the pcr primers were designed to detect the presence of the lkta gene, which ...200919409584
evolution of oxidative/nitrosative stress biomarkers during an open-field vaccination procedure in sheep: effect of melatonin.melatonin has been shown to exert immunomodularory properties with broad application in veterinary medicine. in previous work we have described that subcutaneous coadministration of melatonin to seeps vaccinated against two stumps of a1 and c strains of dichelobacter nodosus enhanced both the antibody titer and serum igg levels to a1 and c strains of d. nodosus compared to vaccinated animals not treated with melatonin. following a similar protocol here we have investigated the effect of a higher ...201019631992
modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (dichelobacter nodosus) vaccines in sheep.virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium dichelobacter nodosus is the essential transmitting agent. commercial multivalent footrot vaccines containing nine fimbrial serogroups (a through i) of d. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. the latter were important compon ...200919853681
footrot and interdigital dermatitis in sheep: farmer satisfaction with current management, their ideal management and sources used to adopt new strategies.the aims of this research were to identify management practices that sheep farmers currently use to treat and prevent footrot in sheep and whether they consider that these are successful management tools and to find out how sheep farmers would ideally like to manage footrot in their flock. over 90% of lameness in sheep in the uk is caused by dichelobacter nodosus, which presents clinically as interdigital dermatitis (id) alone or with separation of hoof horn (fr). a questionnaire was sent to 265 ...201020594602
Displaying items 101 - 200 of 256