A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1ß in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

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 results for diseases transmitted through environmental sources is unclear. In this article a compartmental genetic-epidemiological model was developed to quantify the impact of host genetic diversity on epidemiological characteristics of diseases transmitted through a contaminated environment. The model was parameterized for footrot in sheep. Genetic variation was defined through continuous distributions with varying shape and degree of dispersion for different disease traits. The model predicts a strong impact of genetic heterogeneity on the disease risk and its progression and severity, as well as on observable host phenotypes, when dispersion in key epidemiological parameters is high. The impact of host variation depends on the disease trait for which variation occurs and on environmental conditions affecting pathogen survival. In particular, compared to homogeneous populations with the same average susceptibility, disease risk and severity are substantially higher in populations containing a large proportion of highly susceptible individuals, and the differences are strongest when environmental contamination is low. The implications of our results for the recording and analysis of disease data and for predicting response to selection are discussed.

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 farmers to investigate their current management and their satisfaction with current management of the spectrum of clinical presentations of footrot. Farmers were also asked their ideal management of footrot and their interest in, and sources of information for, change. Approximately 160 farmers responded. Farmers satisfied with current management reported a prevalence of lameness < or = 5%. These farmers caught and treated lame sheep within 3 days of first seeing them lame, and treated sheep with FR and ID with parenteral antibacterials. Farmers dissatisfied with their management reported a prevalence of lameness >5%. These farmers practised routine foot trimming, footbathing and vaccination against footrot. Whilst 89% of farmers said they were satisfied with their management of FR over 34% were interested in changing management. Farmers identified veterinarians as the most influential source for new information. Farmers reported that ideally they would control FR by culling/isolating lame sheep, sourcing replacements from non-lame parents, trimming feet less, using antibacterial treatments less and using vaccination more. Footbathing was a commonly used management that was linked with dissatisfaction and that also was listed highly as an ideal management. Consequently, some of the ideal managements are in agreement with our understanding of disease control (culling and isolation, sourcing healthy replacements) but others are in contrast with our current knowledge of management and farmers self-reporting of satisfaction of management of footrot (less use of antibacterial treatment, more footbathing and vaccination). One explanation for this is the theory of cognitive dissonance where belief follows behaviour, i.e. farmers report that they believe an ideal which is what they are currently doing, even if the management is sub-optimal.

A within farm clinical trial to compare two treatments (parenteral antibacterials and hoof trimming) for sheep lame with footrot.

From observational studies, farmers who use parenteral antibacterials to promptly treat all sheep with footrot (FR) or interdigital dermatitis (ID) have a prevalence of lameness of < 2% compared with a prevalence of 9% lameness reported by farmers who treat lame sheep by trimming affected feet. We tested the hypothesis that prompt treatment of sheep lame with naturally developing FR or ID with parenteral and topical antibacterials reduces the prevalence and incidence of lameness with these conditions compared with less frequent treatment with trimming of hoof horn and applying topical antibacterials.A further hypothesis was that reduction of ID and FR would improve productivity. A lowland sheep flock with 700 ewes was used to test these hypotheses in an 18-month within farm clinical trial with four groups of ewes: two intervention and two control. The duration and severity of lameness was used to categorise sheep into three weighted scores of lameness (WLS): never lame (WLS0), mildly lame/lame for < 6 days (WLS1) and severely or chronically lame (WLS2). The intervention reduced the prevalence of lameness due to FR and ID in ewes and lambs and the incidence of lameness in ewes. The WLS was also significantly lower in sheep in the intervention groups. Ewes with a higher WLS were subsequently significantly more likely to have a body condition score < 2.5 and to have lame lambs. Significantly more ewes lambed and successfully reared more lambs that were ready for slaughter at a younger age in the intervention versus control groups. There was an increase in the gross margin of Pound630/100 ewes mated in the intervention group, including the cost of treatment of Pound150/100 ewes mated. We conclude that prompt parenteral and topical antibacterial treatment of sheep lame with ID and FR reduced the prevalence and incidence of these infectious conditions and led to improved health, welfare and productivity.

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 amplification of a 783 base pairs (bp) product. Serogrouping by multiplex PCR using group (A-I)-specific primers revealed the presence of serogroup B-specific bands of 283 bp.

Effect of parenteral selenium administration to sheep on prevalence and recovery from footrot.

BACKGROUND: Insufficient intake of selenium (Se) is common in many regions, and can contribute to increased susceptibility to and prolonged recovery from infectious diseases. OBJECTIVE: To determine the effect of Se administration in decreasing the severity and prevalence of footrot in sheep. ANIMALS: Thirty-eight footrot-affected and 19 nonaffected sheep from a commercial flock of known high incidence of footrot. METHODS: Placebo-controlled, prospective, 15-month clinical trial. Footrot-affected sheep were randomly assigned into 2 groups (n = 19) and injected with either 5 mg Se (footrot [FR]-Se) or saline (FR-Sal) at 1-month intervals for the duration of the study. Unaffected sheep (controls) received no treatment. Sheep feet were examined, trimmed, and scored for footrot with a scale of 0 (no footrot) to 4 (extensive) at 0, 3, 6, 9, and 15 months. Sheep were also bled at time 0 and then at 3, 6, and 15 months to assess whole blood Se concentrations. RESULTS: At time 0, control sheep (255 +/- 11 ng/mL) had higher (P < .05) whole blood Se concentrations compared with FR-Se (205 +/- 12 ng/mL) and FR-Sal (211 +/- 14 ng/mL) sheep. By 6 months, FR-Se sheep (317 +/- 9 ng/mL) had whole blood Se concentrations greater (P < .05) than both control (281 +/- 14 ng/mL) and FR-Sal (277 +/- 16 ng/mL) sheep. FR-Se ewes showed a faster decline in highest lesion score at 3 (P= .012) and 6 (P= .0036) months, and a greater decrease in the number of feet with foot score >0 at 6 (P= .020) months compared with FR-Sal ewes. Sheep with blood Se concentrations <300 ng/mL were at 3.5 times greater risk (1.1-12.1 confidence interval, odds ratio) for FR, although this relationship was only significant (P= .04) at 6 months of the study. CONCLUSIONS AND CLINICAL IMPORTANCE: In sheep with footrot, improved Se status in conjunction with routine control practices result in more rapid improvement of foot lesions.

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 both zero-1 and continuous data methods. The average prevalence of any score >0 was 15%, and of scores >1 was 12%. The residual correlation between scorers for SUM_FR was 0.87 and between scoring occasions it was also 0.87, indicating high repeatability or agreement both within and between scorers. No significant differences were detected between scorers or between scoring occasions for any of the traits analysed, or different analytical methods used.

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 detected footrot in 19/94 village sheep flocks surveyed. The 19 affected flocks were distributed among nine different administrative districts whereas the villages selected were in 13 of a total of 16 sheep growing districts. The highest within-flock prevalences were among the seven flocks sampled in Bumthang district (mean 20.4%). The prevalence of the disease within flocks was generally much lower in other affected districts and in three districts a single affected animal was identified in the sample of 14 sheep examined in each village. Nationally, footrot prevalence was estimated to be 3.1% (95% CI 2.16-4.04%). There was a positive association between the receipt of animals from NSBC and the presence of footrot. The prevalence of the disease was higher in flocks with a migratory system of management than in those using a sedentary system. The relative risk of there being footrot in a migratory flock was nine-times higher than in a non-migratory flock. Only one strain of Dichelobacter nodosus (serogroup B) was identified among the 234 isolates obtained from the 19 affected flocks. Sheep with footrot healed quickly when treated with a vaccine made from this strain.

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination.

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test for D. nodosus. A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.