Academic literature on the topic 'Dichelobacter nodosus'

The bacterium Dichelobacter nodosus (D. nodosus) is the causative agent of ovine footrot. The aim of this field study was to determine the prevalence of D. nodosus in German sheep flocks. The sheep owners participated voluntarily in the study. More than 9000 sheep from 207 flocks were screened for footrot scores using a Footrot Scoring System from 0 to 5 and sampling each sheep using one interdigital swab for all four feet of the sheep. The detection and discrimination between benign and virulent strains was done employing a real-time PCR. Our results showed a mean prevalence of 42.93% of D. nodosus in German sheep on an animal level. Underrunning of hoof horn on at least one foot (Scores 3-5) was detected in 567 sheep (6.13%). Sheep with four clinically healthy feet were found through visual inspection in 47.85% of all animals included in this study. In total, 1117 swabs from sheep with four clinically healthy feet tested positive for D. nodosus. In 90.35% of the positive swabs, virulent D. nodosus were detected. Benign D. nodosus were detected in 4.74% of the D. nodosus-positive swabs while 4.91% tested positive for both, benign and virulent D. nodosus. In 59 flocks D. nodosus were not detected and in 115 flocks only virulent D. nodosus were found while seven flocks tested positive for benign strains.

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 Fusobacterium necrophorum (40%), Dichelobacter nodosus (31.7%), Porphyromonas asaccharolytica (21.1%) and Prevotella melaninogenica (12.9%). Virulence factors identified in the isolated microorganisms included haemolysins, elastases and lecithinases, which enabled the organisms involved to initiate and/or aggravate the disease. Serotyping was performed for Dichelobacter nodosus isolates, since this species is responsible for triggering the process of infection. A and C were the most frequently isolated serovarieties (representing 40.7% and 25.9% of the cases, respectively).

ABSTRACT 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 (σ54) 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 process that was shown to occur at a PilR- and σ54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and σ54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and σ54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a σ54-dependent PilR/S system that also indirectly controls protease secretion.

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 rapid identification of Gram-negative rod-shaped anaerobes (1997); (c) the API rapid 32 A system (bio Mérieux), and (d) Mast ID™ Anaerobe ID Ring (MID8) (Mast Diagnostics). None of the four methods used allowed us to correctly identify the D. nodosus strains (neither the strains isolated from cases of ovine footrot nor those originating from type collection). Because of the difficulties encountered in obtaining a correct identification of D. nodosus, we propose a simple, rapid and effective way to achieve this task. Our flowchart will provide the means to identify this microorganism in any laboratory of general microbiology without having to use any specialised equipment.

ABSTRACTType IV fimbriae are essential virulence factors ofDichelobacter nodosus, the principal causative agent of ovine foot rot. ThefimAfimbrial subunit gene is required for virulence, butfimAmutants 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 thepilTorpilUgene eliminated the ability to carry out twitching motility. However, thepilTmutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas thepilUmutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway inD. nodosus. It was postulated that sufficient fimbrial retraction must occur in thepilUmutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in apilUmutant ofD. nodosus, aberrant motion was detected in an equivalent mutant ofPseudomonas aeruginosa. These observations explain how inD. nodosusprotease secretion can occur in apilUmutant but not in apilTmutant. In addition, virulence studies with sheep showed that both thepilTandpilUmutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.

Footrot is a contagious hoof disease of ruminants. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. The disease results from a mixed bacterial infection, but the essential agent is Dichelobacter nodosus, a Gram-negative, anaerobic bacterium that possesses type-IV fimbriae on its surface. Genetic variation in the fimbriae of D. nodosus was investigated in this study. Using the polymerase chain reaction (PCR), the variable region of the gene encoding the fimbrial subunit (fimA) was amplified from bacterial DNA extracted from footrot lesions. Different fimA amplimers were differentiated by single-strand conformation polymorphism (SSCP) analysis. In conjunction with DNA sequencing, 15 unique sequences of D. nodosus fimA were obtained from 14 footrot samples taken from 6 farming regions throughout New Zealand. When these sequences were compared to fimA of known serogroups, it revealed that there were at least 15 D. nodosus strains, representing 8 serogroups, present on New Zealand farms. The predominant serogroup was B which contained 6 strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Twelve out of the 15 New Zealand D. nodosus strains had fimbriae different to those previously reported and the presence of multiple strains on a single hoof was common (86% of samples). The fimA sequences from the 12 D. nodosus strains incorporated into the footrot vaccine currently available in New Zealand were determined. A primer set targeting the relatively conserved fimA regions and based on the published sequence of serogroup M Nepalese isolates (designated M-Nep), failed to amplify fimA from the vaccine serotype M strain (designated as M-SPAHL). When the downstream primer was substituted with a primer that was specific for other serogroups of D. nodosus, the fimA gene was successfully amplified. Cloning followed by DNA sequencing, revealed that M-SPAHL fimA was different to M-Nep fimA. The predicted amino acid sequence of M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not M-Nep. The sequence difference between M-SPAHL and M-Nep was larger than that expected within a serogroup. The consequences of serological relatedness and sequence dissimilarity are discussed. Only eight of the 15 New Zealand field strains had fimbriae identical to those of the vaccine strains, while the remaining seven strains possessed different fimbriae. In addition, the vaccine contained two more D. nodosus strains, representing two sera groups, that were not found on the New Zealand farms investigated in this study. This may, to some extent, explain why the current footrot vaccine is at times less efficient in New Zealand. Another 17 footrot samples were screened for new or additional D. nodosus strains. Two PCR amplimers (designated X and Y) derived from footrot samples generated SSCP patterns different to those of previously identified strains. DNA sequencing revealed that these two fragments possessed novel sequences. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1 whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M1 or E1, produced PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M1 and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artefacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It therefore seems more likely that fragments X and Y reflect genuine fimA sequences of D. nodosus which have resulted from in vivo DNA recombination, than from a PCR recombination artifact. The genetic capability for recombination at the fimbrial subunit locus may therefore endow D. nodosus with the ability to alter its antigenic appearance. D. nodosus strains present in footrot lesions can be genotyped using a PCR-SSCP/sequencing technique. However, this typing technique requires cloning and screening of D. nodosus fimA sequences, which is both laborious and costly. A rapid molecular typing system for D. nodosus was therefore developed in this study. A close examination of available D. nodosus fimA sequences revealed regions that appear to be specific for serogroups and serotypes. These regions were used to design a panel of sequence-specific oligonucleotide probes (SSOPs), and a rapid and accurate D. nodosus typing system using PCR and reverse dot-blot hybridisation (PCR/oligotyping) was subsequently developed. 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 mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments that had hybridised were detected. The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing, was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types were present in the original footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus present in a footrot sample can be accurately genotyped in less than two days. Individual animals from the same farm, or the same paddock, were often infected by different strains of D. nodosus. This suggests a host role in mediating footrot infection, or that the interaction between the pathogen and the host is important. In order to better understand the interaction between the bacterium and the host, two polymorphic ovine class II MHC genes DQA1 and DQA2, which have been previously shown to be important in footrot infection, were also investigated in this study. PCR-SSCP/sequencing analysis of the DQA1 locus revealed ten unique ovine DQA1 sequences, with five of them being newly identified. This increases the number of known ovine DQA1 alleles from 8 to 13 (including a null allele), implying a high level of polymorphism at the ovine DQA1 locus. D. nodosus present on 20 footrot infected sheep from the same flock were genotyped, together with the ovine DQA1 and DQA2 genotypes of their hosts. Preliminary results showed that sheep with the same DQA1 and DQA2 genotypes tended to be infected by similar types of D. nodosus. Different types of D. nodosus were generally found on sheep with different genotypes at either the DQA1 or the DQA2 locus. This suggests the diversity in D. nodosus infection may be associated with the heterogeneity in the host MHC. However, as only a small number of animals from the same sire were analysed, further investigation is needed to gain a better understanding of the interaction between D. nodosus and the host MHC.

Ovine footrot (FR) is an economically important disease that causes lameness and affects sheep flocks worldwide. It is characterized by interdigital skin inflammation (interdigital dermatitis [ID]) with, or without, separation of the hoof horn from the underlying tissue (severe footrot [SFR]). The primary causative agent is the gram-negative anaerobic bacterium Dichelobacter nodosus, which is present in diseased feet and thought to be transmitted via contaminated surfaces. Periods of apparent zero prevalence of FR in a flock can be followed by disease occurrence when the climate becomes favourable for pathogen transmission. This suggests that there are sites where D. nodosus persists in the absence of disease. These sites might include healthy feet, the gingival cavity and faeces of sheep and also the environment. The aim of this thesis was to investigate persistence of D. nodosus, by investigating possible sites of survival of D. nodosus over time. Prospective longitudinal studies were used to investigate persistence. Samples were collected from sheep and from the pasture in three studies (Studies 1 and 2: England, study 3: Spain). Quantitative PCR was used to detect and quantify D. nodosus and to investigate associations between D. nodosus presence in feet, in the gingival cavity and on pasture and a range of predictor variables including climate. A multiple locus variable number tandem repeat analysis (MLVA) suitable for use on mixed DNA and environmental samples was optimized and validated to investigate D. nodosus strains within and between sites. A novel approach to characterize individual strains in a sample was designed. D. nodosus was detected in all sample types in all studies but not on all occasions. The feet of sheep were the only site where D. nodosus was detected in loads exceeding 103 cells per swab. In study 1, D. nodosus was detected in amounts exceeding103 cells in samples collected from the pasture in week 1 only, when detection frequency of D. nodosus on feet was high and the weather was wet. A minimum of 14 strains of D. nodosus were detected on the feet of sheep by MLVA. A decline in detection of D. nodosus in the environment coincided with periods of dry weather, however, dry weather did not coincide with a decline in D. nodosus loads on feet or incidence of disease. D. nodosus was more likely to be detected in the gingival cavity when a sheep had FR. It was detected in 25 % of gingival cavity samples and strain types identified in the gingival cavity were the same as the dominant strain types on the feet of sheep. In study 2, disease prevalence and D. nodosus detection frequencies were lower than in study 1. When sheep from the study group were separated from the main flock in week 1 and moved onto pasture that had been unoccupied for 10 days, D. nodosus was transferred to the study group on healthy feet. One dominant strain of D. nodosus persisted throughout an episode of disease and this strain was present on the healthy feet of sheep until up to 5 weeks before the development of lesions in high bacterial loads. There was a reduction in lesion severity and reduced detection of D. nodosus in soil in a period of dry weather. Only 1 sample from the gingival cavity was positive for D. nodosus. Two faecal sample were positive for D. nodosus, indicating for the first time that faecal shedding is possible. In study 3, there were high loads of D. nodosus on healthy feet of a sheep that was classed as susceptible when there had been no cases of FR for at least 2 month. D. nodosus was still present in the flock during the long non-transmission period in the summer. We conclude that D. nodosus is more likely to persist on the feet of sheep, whereas long-term environmental reservoirs of D. nodosus are unlikely. Future research should focus on the feet of sheep and possibly faeces as possible sites of persistence of D. nodosus in the absence of disease.

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Doenças podais são uma das principais injúrias em rebanhos de pequenos ruminantes em diversos países e a pododermatite infecciosa (Footrot) é relatada como a mais frequente em ovinos no Brasil. No Rio Grande do Sul, as doenças podais ainda são um grave problema para os criadores de ovinos e pouco tem sido feito para saná-las. O Footrot, mesmo se tratando de uma doença de notificação obrigatória e frequente na região, os registros oficiais sobre a situação da doença nos rebanhos são escassos. Esse trabalho teve como objetivo descrever as principais características das lesões podais observadas em ovinos da Mesorregião Sudoeste do Rio Grande do Sul, com ênfase nos aspectos epidemiológicos, macroscópicos, microscópicos e radiográficos das lesões de Footrot. O estudo foi realizado em duas etapas. Inicialmente foram avaliados ovinos em 27 propriedades rurais, das quais 21 registraram a ocorrência de lesões podais em ovinos com perdas econômicas significativas. Aproximadamente 1.700 ovinos, em média 10% dos animais do rebanho, apresentavam diferentes graus de claudicação decorrente de lesões podais que, macroscopicamente, variavam de brandas a severas. Posteriormente, foram avaliados os variados graus de lesões de Footrot nos ovinos. Em casos de abate e necropsia, os cascos dos ovinos com as lesões foram submetidos à avaliação macroscópica, radiográfica e microscópica. Dessa forma a doença foi classificada em cinco graus de severidade que variaram de 1 (lesões leves) a 5 (lesões graves). Verificou-se que diversos fatores como clima e manejo foram favoráveis para o desenvolvimento das lesões podais e essas estão associadas, na maioria dos casos, a Footrot em diferentes estágios de evolução. A partir dessa classificação em graus foi possível classificá-los macroscopicamente em duas síndromes clínicas propostas, a saber Footrot benigno e maligno. Essa classificação facilita o estabelecimento das medidas de controle com intuito de limitar a propagação da doença e evitar a evolução das lesões nos cascos acometidos.
Foot diseases are one of the main disorders in small ruminant flocks in several countries and infectious pododermatite (Footrot) is reported as the most frequent podal lesions in sheep in Brazil. In Rio Grande do Sul state, foot diseases still a serious problem for sheep farmers and little has been done to remedy them. Footrot is a notifiable disease and frequent in the region, the official records on the disease situation in herds are scarce. This study aimed to describe the main features of foot lesions observed in sheep from Mesoregion Southwest of Rio Grande do Sul, with emphasis on epidemiology, macroscopic, microscopic and radiographic changes of Footrot injuries. The study was conducted in two steps. Initially, sheep were evaluated on 27 farms, of which 21 showed records of the occurrence of foot lesions in sheep and significant economic losses. Approximately 1,700 sheep, about 10% of the flocks, showed varying degrees of lameness due to foot lesions, macroscopically characterized as mild to severe. Subsequently, they assessed the varying degrees of injuries Footrot in sheep. Hooves with injuries were submitted to macroscopic, radiographic and microscopic evaluation. Thus the disease was classified into five grades of severity ranging from 1 (mild injury) to 5 (severe damage). It has been found that several factors such as weather and handling were favorable for the development of foot injuries and these are associated in most Footrot cases in different stages of evolution. Based on the classification in degrees of infectious pododermatitis it was possible to classify them macroscopically in clinical syndromes proposed as benign and malignant Footrot. This classification facilitates the establishment of control measures with the intention of reduce spread of disease and prevent the development of lesions in affected hooves.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

A peeira é uma doença bastante contagiosa e corresponde à principal causa de claudicação em ovinos. A esta doença é atribuída enorme relevância do ponto de vista económico e de bem-estar animal. Apesar de já ter sido amplamente estudada por todo o mundo, as publicações sobre a doença no Alentejo são escassas. Assim, os principais objetivos deste trabalho foram identificar fatores de risco para a doença nesta região e caracterizar a existência de lesões em rebanhos de ovinos. A aplicação dos Inquéritos de Validação permitiu aferir que os produtores de ovinos possuem elevada capacidade de distinguir as lesões de peeira de outras afeções podais. Os Inquéritos Epidemiológicos aplicados demonstraram uma prevalência estimada de 34,5% para a peeira. A distribuição da doença não é regionalmente homogénea, apresentando valores superiores no Alto Alentejo. A análise dos resultados permitiu identificar vários fatores de risco, onde se destacam: maior dimensão do efetivo; concentração das épocas de partos; pastoreio em áreas de baixa altitude e com fraca capacidade de drenagem. Relativamente às opções terapêuticas, a utilização do pedilúvio e da antibioterapia tópica estão associadas a uma maior proporção de animais afetados, enquanto a separação dos animais doentes exibiu efeito protetor. A análise bioclimática revelou que a pluviosidade constitui o principal fator de risco para a doença. A classificação lesional realizada nos ovinos confirmou a natureza dinâmica da peeira. De facto, a proporção de animais afetados em cada efetivo foi bastante variável, estando dependente da precipitação registada antes de cada visita. Os animais que se revelaram mais resistentes à doença foram as fêmeas, os mais jovens e os de raça merina preta. Nas lesões mais severas registou-se uma maior probabilidade de deteção dos agentes D. nodosus e F. necrophorum, sendo-lhes atribuído o papel de precursor da doença e de agente oportunista secundário, respetivamente; Title: Characterization of ovine footrot in the Alentejo region Abstract: Footrot is a very contagious disease, representing the main cause of lameness in sheep. It is attributed a huge economic and animal welfare impact to footrot. Although it has been widely studied all over the world, research in Alentejo has been scarce. Thus, the main purposes of this study were to perform the identification of risk factors associated with the disease and to characterize the existence of foot lesions in sheep flocks of Alentejo. The implementation of Validation Surveys has enabled us to conclude that sheep producers in Alentejo have a globally high capacity to differentiate footrot lesion from other hoof disorders. Epidemiological surveys applied to a significant number of flocks revealed an estimated prevalence of 34.5%. Even though, the distribution of the disease is not similar across the whole region, exhibiting higher values in Alto Alentejo. The results analysis made it possible to identify several risk factors, where we can highlight: greater size of the sheep flock; concentration of lambing periods; grazing in lowland areas and with weak drainage capability. Regarding therapeutics, the use of footbath or topical antibiotics increased the proportion of affected animals, while the separation of sick animals exhibited a protective effect. Bioclimatic analysis revealed that precipitation is the main risk factor to footrot. The categories of foot scores carried out in sheep confirmed the dynamic nature of footrot. The proportion of animals affected in each herd was quite variable, being highly dependent on the rainfall recorded before each classification period. The animals that proved to be more resistant to disease were females, young and the ones belonging to merina preta breed. The most severe lesions revealed an increasing possibility of detecting bacteria D. nodosus and F. necrophorum, being attributed the roles of disease precursor and secondary opportunist, respectively to both agents.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Studies presented in this thesis looked at developing new methods for the diagnosis of virulent footrot (VFR) in sheep and identification of serogroups of Dichelobacter nodosus, the principal causative agent of footrot. Earlier studies had shown that immunological memory response in sheep recovered from VFR can be aroused by natural or recurrent infection or by injection of outer membrane protein (OMP) antigens to be used as a retrospective diagnostic test for VFR. But OMP antigen was found to be non-specific in older animals. To overcome this non-specificity of OMP antigen in anamnestic response, pilus antigen was evaluated in a trial at Camden. The results of this trial indicated that antibodies to pilus antigen can be detected over time in a manner similar to OMP antibodies so a retrospective assessment of VFR status can be made by anamnestic test with pilus antigens. The anamnestic response to pilus was similar in character to OMP antigen but unlike OMP was highly specific. The response to anamnestic challenge with pilus was determined by severity of the lesions they had expressed, with severe lesions triggering the greater responses. However, there was variation between individuals, with some (6 of 46 with severe lesions) failing to respond. This individual variation is probably mediated genetically as is response to vaccination. This anamnestic test was tested in flocks of sheep in Nepal that had a history of VFR which had apparently been eradicated. That assessment, based on clinical findings, was confirmed by the uniformly negative results in the pilus anamnestic test applied to a representative sample of the population. This allowed a conclusion that the virulent strains of D. nodosus involved had been eliminated from these flocks. As mentioned in the preceding study pilus antigen was found to be very specific and ideal for retrospective diagnosis of virulent footrot with an anamnestic challenge ELISA test. However, serogroup specificity was seen as a disadvantage of using pilus antigen for the anamnestic test. The possibility of using multivalent pilus antigens was tested in another trial. These animals had been involved in a clinical expression experiment conducted by another research group and had a clinical and bacteriological history extending over more than 12 months. After these initial trials all these animals were treated for footrot and managed for 5 months as a single flock at Camden. These were then challenged with multivalent pilus antigen (serogroup A - I) as a single injection. The results obtained indicate that multivalent pilus anamnestic ELISA is equally effective as monovalent pilus. This has the added advantage that prior knowledge of the serogroups present in the flock is not required. It has the possibility of being used as an indirect test to check the presence of serogroups in a flock without doing the bacterial cultures. This test can identify most animals with pre-existing underrunning lesions (Scores of 3 or higher). However, the sensitivity and specificity of this test need to be tested extensively in flocks of known clinical history before it could be adopted as a routine test. As a key component of a larger study to determine the role of fimbrial genes (fimA and fimB) of D. nodosus in the pathogenesis of footrot using allelic exchange to disrupt these genes of a strain (serogroup G), the study presented in this thesis contributed a detailed characterisation of the resultant mutant and the wild strains and tested these strains for virulence in sheep. The results presented provided the first definitive evidence that the fimA gene is essential for virulence of D.nodosus in sheep. In vivo virulence testing of two fimA mutants showed that they were not able to establish any footrot whereas the wild type of the same strain produced virulent footrot in the same trial conducted under similar conditions. These mutant bacteria were not re-isolated from interdigital skin after in vivo challenge. This indicated that fimA mutant strains could not colonise the ovine foot, and the simplest and most likely explanation for these results was that colonisation of the interdigital skin and subsequent penetration of the stratum corneum requires the adhesive activity of type IV fimbriae. However, since these mutants also had altered ability to secrete extracellular proteases, and perhaps other as yet unknown extracellular proteins, the possibility of the involvement of these factors as major determinants of host colonisation or invasion cannot be excluded. Current methods for the identification of the serogroup of D. nodosus present in the bacterial population requires isolation of the organism and after purification by subculture, antigenic analysis with agglutination tests. This usually takes at least 3 to 4 weeks. With the objective of developing a rapid serogroup specific PCR assay, the basis of serogroup variation in D. nodosus localised in the fimbrial gene region was exploited. A common forward primer and 9 serogroup specific reverse primers were selected from the fimbrial gene sequences of the prototype strains. Analytical sensitivity of the serogroup specific primers on chromosomal DNA was similar to PCR tests in other bacterial species reported before. Immuno-magnetic bead capture PCR method was able to detect 5 to 10 cells in cell lysates. Specificity within and between the serogroups of D. nodosus was tested with all the prototype strains. They were found to be very specific to each serogroup and specific only to D. nodosus when tested with 84 commonly found bacterial strains or strains related to D. nodosus. To overcome the time delay in conducting 9 different amplifications to find out the prevalence of all possible serogroups in a flock multiplex PCR reactions with common forward primer and groups of 3, 4 and 5 reverse primers were successful in reducing the number of reactions to 2 (with groups of 4 and 5) or 3 (with groups of 3) primers. A drawback of the multiplex reaction was that if a template was 1000 times less concentrated that the others in the mixture it was not amplified but the margin for difference is very high. The main aim of developing rapid serogroup specific PCR was to apply these tests directly on footrot lesion samples to make it a rapid diagnostic test for field samples. The sensitivity of the test on lesion samples was found to be very low. To try and improve the sensitivity an overnight or four days old pre-enrichment culture in broth was tested but was found to be no better than direct PCR. The immuno-magnetic capture method which improved the sensitivity of pure culture samples by 10 -100 fold also had very low sensitivity with lesion samples. However, this drawback can be overcome by picking up colonies from 4 days old lesion cultures on hoof agar (HA) plates for serogroup specific multiplex PCR. If the colonies are too small/ too few on the lesion cultures these can be sub cultured onto a quarter of 4 percent HA plates and then used for the PCR test which also reduces the time taken for serogrouping at least by 2 weeks. The other advantage is that individual colonies do not need to be isolated. A PCR test can be done on pooled colonies just as well and can be used to identify all serogroups present in the sample. Serogroup specific PCR is much faster and is more sensitive and accurate than slide agglutination tests which take 3 to 4 weeks to complete. Multiplex PCR makes it easier to detect different serogroups in a sample with a maximum of 3 PCR tests. Serogroup specific multiplex PCR will be a useful tool for footrot control based on specific vaccination. The difficulty in using the test on direct lesion swabs needs to be further looked into. There may be future advances in the application of PCR tests to clinical samples.

Ovine footrot is an infectious disease of sheep which causes serious economic losses to countries worldwide and costs the UK £84 M per annum. For effective control of this disease which is a major animal welfare issue, it is essential to study mechanisms of transmission. One of the key aims of the project was to improve our understanding of the potential for Dichelobacter nodosus, the causal agent, transmission via the environment within a flock of ewes and lambs. Newborn lambs were free from D. nodosus presence on their feet, but rapidly became contaminated with D. nodosus within 5-13 h after birth as detected using the specific molecular markers rpoD and 16S rRNA gene. The likely source of contamination of lambs was the straw bedding from the communal pens. A diverse population of D. nodosus was observed on the feet of ewes and lambs as determined by the presence of multiple strains with variable numbers of pgrA tandem repeats in the R1 region. This was further supported by Multi Locus Variable Number Tandem Repeats (MLVA) typing of the isolates, which also indicated high variation in the alleles present on the ewes and lambs. The primary aim was to determine if multiple strains present on the ewes were vertically transmitted to the lambs. This work has clearly demonstrated that no vertical transmission occurred between ewes and lambs and some strains, but not all were shared between ewe and its lamb suggesting transmission from their mother or other ewes sharing the same lambing pen. D. nodosus was detected in a range of environmental samples such as bedding, faecal balls compacted within the interdigital space and soil suggesting that shedding into the environment is the main route of D. nodosus transmission. Survival studies provided evidence that the pathogen persisted in soil microcosms for at least 40 days with viable cells persisting for a minimum of 30 days in four soil types. A lower temperature of 5 oC and clay soil was associated with longer duration of survival. Single Nucleotide Polymorphism (SNP) analysis of isolates from the ewes and lambs indicated that two main clonal populations existed that represented two clusters α and β within clade I, a virulent clade from the recent genome study including 103 global D. nodosus strains. No UK strains grouped with the benign clade II. Previous diversity studies on the isolates indicated diversity in MLVA types, pgr alleles and pgrA tandem repeats, but these did not correlate with the clustering; clusters α and β contained a mix of pgrA and pgrB but both were in clade I. This conflicts with the role of pgrB as a non-virulent allele although pgrA expression was induced by hoof horn in vitro, whereas pgrB was not. The work reported in this thesis has improved our understanding of the environmental transmission of D. nodosus between sheep, longevity of the pathogen in soil and diversity of strains in the UK.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020
A Peeira é uma doença que afecta ovinos e outros ruminantes, sendo atualmente uma das principais causas de claudicação em ovelhas. Tem como principal agente patogénico a bactéria gram-negativa anaeróbica Dichelobacter nodosus. Esta doença tem grandes impactos económicos e relevância em termos de bem-estar animal, tornando-se por isso essencial o seu controlo, tratamento e erradicação. Existem essencialmente duas manifestações clínicas desta doença. A manifestação ligeira consiste na inflamação da epiderme interdigital, sendo por essa razão denominada Dermatite Interdigital (ID – Interdigital Dermatitis). Por outro lado, na manifestação mais grave, esta inflamação pode progredir e causar a separação do casco e do tecido subjacente, sendo por essa razão chamada de Peeira Grave (SFR – Severe footrot). A severidade desta doença está principalmente dependente de três fatores: a virulência da estirpe infectante de D. nodosus, as condições ambientais e a resistência natural do hospedeiro. Alguns dos fatores ambientais que contribuem para a progressão e transmissão da doença são a temperatura, a humidade e o tipo de solo. Foi também demonstrado que a coinfecção com bactérias de outros géneros desempenha um papel deveras importante no desenvolvimento e progressão da doença. Algumas das bactérias oportunistas que foram identificadas na pele interdigital de ovinos com Peeira são Fusobacterium necrophorum e Treponema spp.. O papel exercido por estas bactérias oportunistas na progressão da doença ainda não é totalmente compreendido. Verificou-se, no entanto, que F. necrophorum, também uma bactéria gram-negativa anaeróbica, contribui para o desenvolvimento e progressão da peeira e que exerce uma relação sinergística com D. nodosus, tornando esta bactéria oportunista um foco central no estudo da doença. Em relação à virulência de D. nodosus, os dois factores principais reconhecidos são as fímbrias do tipo IV e as proteases extracelulares. A análise das fímbrias permite a discriminação de dez serogrupos (A-I e M) com base na sua diversidade estrutural que é causada por variações na região carboxi-terminal da subunidade fimbrial, também chamada proteína FimA e codificada pelo gene fimA. A protease acídica 2 (AprB2/V2), em particular, é um importante fator de virulência responsável pela atividade de elastase presente nas estirpes virulentas que, por sua vez, é essencial para o desenvolvimento das lesões características da Peeira. Os genes aprV2 e aprB2 diferem apenas numa substituição de dois pares de base (TA/CG) na posição 661/662, o que resulta na alteração de um aminoácido (Tyr92Arg) na proteína madura, sendo que esta alteração determina a atividade de elastase. O principal objetivo do presente estudo foi determinar a prevalência de D. nodosus e F. necrophorum em ovelhas da região do Alentejo com diferentes manifestações de Peeira e isolar e caracterizar D. nodosus quanto à sua virulência e serogrupo. Foram utilizadas amostras de tecido da epiderme interdigital provenientes de ovelhas saudáveis (grau 0) e com Peeira (graus 1-5) de 17 explorações de 13 concelhos da região do Alentejo. Amostras de DNA das biópsias recolhidas (n=261) foram utilizadas para a deteção e identificação de D. nodosus e F. necrophorum e para determinar a virulência e os serogrupos de D. nodosus. Estes testes foram realizados através de PCR em tempo real. Os fatores de virulência foram ainda caraterizados com recurso a sequenciação de Sanger. A determinação dos serogrupos foi realizada por PCR em formato multiplex. O isolamento de D. nodosus a partir de amostras de biópsia foi realizado por cultura bacteriológica em meios seletivos. Os resultados deste estudo mostraram que, na região do Alentejo, existe uma prevalência de D. nodosus (51%, n= 132) e F. necrophorum (46,4%, n=121) elevada em casos clínicos de Peeira. Verificou-se também uma associação entre a prevalência destes dois agentes e a severidade da lesão, ou seja, com a progressão da doença para fases mais avançadas. D. nodosus não foi detetado em quatro das 17 explorações em estudo enquanto F. necrophorum apenas estava ausente numa dessas explorações. Observou-se ainda uma tendência para explorações com um maior número de animais infetados por D. nodosus também apresentarem uma elevada prevalência de F. necrophorum. A coinfecção por ambas as espécies é a situação epidemiológica mais comum, estando associada a uma maior severidade da doença, quando comparada com as situações em que se registou infeção apenas com D. nodosus. Em relação à prevalência geográfica de D. nodosus, este foi detetado em 11 dos 13 concelhos, não se tendo detetado no Alandroal e Moura. As prevalências mais elevadas foram registadas em Almodôvar (83%), Portel (72,4%) e Évora (68%). A análise dos fatores de virulência nas amostras de DNA de biópsia onde D. nodosus foi detetado (n=132) revelou que a maioria dos isolados (n=127) era virulenta, com exceção de cinco cujo resultado foi indeterminado. Com base em critérios espaciais e qualitativos, das 132 amostras de DNA de biópsia positivas para a presença de D. nodosus foram selecionadas 53 para a determinação dos serogrupos, tendo-se confirmado o serogrupo em apenas 19 dos casos, distribuídos nas seguintes proporções: serogrupo B (90%), serogrupo C (5%) e serogrupo F (5%). Quanto à cultura bacteriológica, das 132 amostras processadas onde foi detetado D. nodosus foram obtidos 17 isolados identificados como D. nodosus por PCR em tempo real, tendo estes sido caraterizados quanto à virulência e serogrupo a partir de DNA extraído de culturas puras. Com a exceção de um isolado, os resultados dos testes de virulência foram concordantes com os resultados obtidos nas amostras de DNA de biópsia, confirmando os isolados como virulentos. O serogrupo de 15 dos 17 isolados foi identificado, tendo dois permanecido indeterminados. Os 15 isolados pertenciam a seis diferentes serogrupos, B, C, F, G, D e H, sendo que a maioria dos isolados pertencia ao serogrupo B (40%). O segundo serogrupo mais comum foi o serogrupo C (20%), seguindo-se, na mesma proporção, os serogrupos H e G (13%). Combinando os resultados provenientes da análise por PCR multiplex de DNA de biópsia total e de DNA de culturas puras, determinou-se os serogrupos de 29 (22%) das 132 amostras de ovelhas positivas para D. nodosus. Estes 29 serogrupos foram identificados em animais de explorações de oito dos 11 concelhos positivos para D. nodosus (73%), tendo o serogrupo B sido detetado em sete concelhos com a exceção de Odemira. Em contraste, os serogrupos D e H só foram encontrados num concelho cada (Portel e Ponte de Sôr, respectivamente). Em relação à localização geográfica, é importante mencionar a elevada prevalência em concelhos geograficamente próximos, incluindo o concelho de Évora (68%), Portel (72,4%) e Alvito (65,4%). Dois outros municípios com elevada prevalência são Almodôvar (83%) e Odemira (67%), os concelhos mais a sul incluídos neste estudo e também geograficamente próximos. Interessantemente, os três concelhos com menor prevalência aparente, Alandroal (0%), Moura (0%) e Serpa (20%), situam-se perto da fronteira com a Espanha. O presente estudo parece indicar que, na região do Alentejo, existe uma elevada prevalência de D. nodosus, assim como de F. necrophorum, em casos clínicos de peeira. Confirmou-se uma relação entre as duas espécies bacterianas, sendo que a infeção por D. nodosus é influenciada pela presença de F. necrophorum. Os resultados obtidos sugerem ainda que esta espécie bacteriana atua como agente patogénico secundário, aumentando a severidade das lesões e estando envolvida predominantemente nas fases mais tardias da Peeira. Conclui-se também que as estirpes de D. nodosus presentes nesta região de Portugal são maioritariamente estirpes virulentas e que os serogrupos mais comuns são os serogrupos B e C. Verificou-se ainda que poderão existir possíveis “hotspots” de infeção por D. nodosus nas zonas em torno do concelho de Évora e no sul do Alentejo, junto aos concelhos de Almodôvar e de Odemira. Esta investigação consiste no segundo estudo realizado em Portugal com foco na caracterização da Peeira e no primeiro com a utilização de métodos atualizados de diagnóstico e caracterização molecular de D. nodosus (PCR em tempo real e PCR multiplex). Os resultados desta dissertação salientam a importância do estudo da Peeira em Portugal, tendo em consideração a sua elevada prevalência e a virulência das estirpes de D. nodosus. A identificação dos serogrupos predominantes e a caraterização dos isolados neste estudo epidemiológico é um primeiro passo para um melhor controlo e possível erradicação da doença no país, nomeadamente através da implementação de medidasimunoprofiláticas e medidas de biossegurança nas explorações afetadas e noutras que, com estas, mantêm relações comerciais.