Survival of the ovine footrot pathogen Dichelobacter nodosus in different soils.

Dichelobacter nodosus (D. nodosus) is the causative agent of footrot in sheep; one of the most important health and welfare issues of sheep worldwide. For control programmes to be effective, it is essential that the transmission cycle of D. nodosus is understood and bacterial reservoirs in the environment are better defined. This study evaluated the survival of D. nodosus in different soils using soil microcosms. Cultivation independent and dependent methods were used to detect D. nodosus over 40 days from seeding in soil. A D. nodosus specific probe was used for quantification by qPCR and viability was assessed by cell permeability to an intercalating dye, PMA, and by culture. Survival varied dramatically depending on soil type, matric potential (MP) and temperature. Our findings indicate that D. nodosus survival was higher at 5 °C compared with 25 °C in all soils and significantly longer at both temperatures in clay soil (>44% clay) compared with other soil types. Survival under all conditions was longer than 30 days for both culture independent and dependent methods, this is substantially longer than previous studies and, if this is an infectious dose, longer than the current recommendation of resting a field for 14 days to prevent onward infection.

Sand fly synthetic sex-aggregation pheromone co-located with insecticide reduces the incidence of infection in the canine reservoir of visceral leishmaniasis: A stratified cluster randomised trial.

OBJECTIVE: To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. METHODS: A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. RESULTS: A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. CONCLUSION: The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

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 was detected 5-13 h after birth, once they had stood on bedding containing naturally occurring D. nodosus. Multiple genotypes identified by cloning and sequencing a marker gene, pgrA, and by multi locus variable number tandem repeat analysis (MLVA) of community DNA from swabs on individual feet indicated a mixed population of D. nodosus was present on the feet of both ewes and lambs. There was high variation in pgrA tandem repeat number (between 3 and 21 repeats), and multiple MLVA types. The overall similarity index between the populations on ewes and lambs was 0.45, indicating moderate overlap. Mother offspring pairs shared some alleles but not all, suggesting lambs were infected from sources(s) other than just their mother's feet. We hypothesise that D. nodosus is transferred to the feet of lambs via bedding containing naturally occurring populations of D. nodosus, probably as a result of transfer from the feet of the group of housed ewes. The results support the hypothesis that the environment plays a key role in the transmission of D. nodosus between ewes and lambs.

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 agent of ovine FR, however, the role of Fusobacterium necrophorum and other bacteria present in the environment and on the feet of sheep is less clear. The objective of this study was to use fluorescence in situ hybridisation (FISH) to detect, localise and quantify D. nodosus, F. necrophorum and the domain Bacteria from interdigital skin biopsies of healthy, ID- and SFR-affected feet. D. nodosus and F. necrophorum populations were restricted primarily to the epidermis, but both were detected more frequently in feet with ID or SFR than in healthy feet. D. nodosus cell counts were significantly higher in feet with ID and SFR (p<0.05) than healthy feet, whereas F. necrophorum cell counts were significantly higher only in feet with SFR (p<0.05) than healthy feet. These results, together with other published data, indicate that D. nodosus likely drives pathogenesis of footrot from initiation of ID to SFR; with D. nodosus cell counts increasing prior to onset of ID and SFR. In contrast, F. necrophorum cell counts increase after SFR onset, which may suggest an accessory role in disease pathogenesis, possibly contributing to the severity and duration of SFR.

A longitudinal study of the role of Dichelobacter nodosus and Fusobacterium necrophorum load in initiation and severity of footrot in sheep.

Footrot is an infectious bacterial disease of sheep that causes lameness. The causal agent is Dichelobacter nodosus. There is debate regarding the role of Fusobacterium necrophorum in disease initiation. This research used an observational longitudinal study of footrot, together with quantitative PCR (qPCR) of bacterial load of D. nodosus and F. necrophorum, to elucidate the roles of each species in the development of disease. All feet of 18 a priori selected sheep were monitored for five weeks assessing disease severity (healthy, interdigital dermatitis (ID) and severe footrot (SFR)) and bacterial load. A multinomial model was used to analyse these data. Key unadjusted results were that D. nodosus was detected more frequently on feet with ID, whereas F. necrophorum was detected more frequently on feet with SFR. In the multinomial model, ID was associated with increasing log10 load of D. nodosus the week of observation (OR=1.28 (95% CI=1.08-1.53)) and the week prior to development of ID (OR=1.20 (95% CI=1.01-1.42). There was no association between log10 load(2) of F. necrophorum and presence of ID (OR=0.99 (95% CI=0.96-1.02))). SFR was associated with increasing log10 load of D. nodosus the week before disease onset (OR=1.42 (95% CI=1.02-1.96)) but not once SFR had occurred. SFR was positively associated with log10 load(2) of F. necrophorum once disease was present (OR=1.06 (95% CI=1.01-1.11)). In summary, there was an increased risk of increasing D. nodosus load the week prior to development of ID and SFR and during an episode of ID. In contrast, F. necrophorum load was not associated with ID before or during an episode, and was only associated with SFR once present. These results contribute to our understanding of the epidemiology of footrot and highlight that D. nodosus load plays the primary role in disease initiation and progression, with F. necrophorum load playing a secondary role. Further studies in more flocks and climates would be useful to confirm these findings. This study identifies that D. nodosus load is highest during ID. This supports previous epidemiological findings, which demonstrate that controlling ID is the most effective management strategy to prevent new cases of ID and SFR.

Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics.

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool.

Detection and diversity of a putative novel heterogeneous polymorphic proline-glycine repeat (Pgr) protein in the footrot pathogen Dichelobacter nodosus.

Dichelobacter nodosus, a Gram-negative anaerobic bacterium, is the essential causative agent of footrot in sheep. Currently, depending on the clinical presentation in the field, footrot is described as benign or virulent; D. nodosus strains have also been classified as benign or virulent, but this designation is not always consistent with clinical disease. The aim of this study was to determine the diversity of the pgr gene, which encodes a putative proline-glycine repeat protein (Pgr). The pgr gene was present in all 100 isolates of D. nodosus that were examined and, based on sequence analysis had two variants, pgrA and pgrB. In pgrA, there were two coding tandem repeat regions, R1 and R2: different strains had variable numbers of repeats within these regions. The R1 and R2 were absent from pgrB. Both variants were present in strains from Australia, Sweden and the UK, however, only pgrB was detected in isolates from Western Australia. The pgrA gene was detected in D. nodosus from tissue samples from two flocks in the UK with virulent footrot and only pgrB from a flock with no virulent or benign footrot for >10 years. Bioinformatic analysis of the putative PgrA protein indicated that it contained a collagen-like cell surface anchor motif. These results suggest that the pgr gene may be a useful molecular marker for epidemiological studies.

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 (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.

Differences in microbial activity and microbial populations of peat associated with suppression of damping-off disease caused by Pythium sylvaticum.

The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

Molecular characterization of Legionella populations present within slow sand filters used for fungal plant pathogen suppression in horticultural crops.

The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water.

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.