Bovine Digital Dermatitis: Treponema spp. on trimming equipment and chutes - effect of washing and disinfection.

BACKGROUND: Digital dermatitis (DD) is a contagious bovine foot disease causing reduced animal welfare and negative economic consequences for the farmer. Treponema spp. are the most important causative agents. Studies indicate that trimming equipment can transfer DD-associated treponemes between cows. The aim of this observational study in 22 DD-positive Norwegian dairy herds was to investigate the risk of transferring Treponema spp. with trimming equipment and chutes after claw trimming, and after washing and disinfection. Swabs from the trimming equipment and chutes were collected from nine different locations, at five different time points. Bacterial DNA was extracted from 647 swabs and analysed by qPCR for Treponema spp. In addition, 172 swabs taken immediately after trimming, were analysed by a multiplex qPCR targeting T. phagedenis, T. pedis and T. medium/vincentii. Biopsy sampling from DD lesions was performed on cows in the same herds during trimming. Altogether 109 biopsies were analysed by FISH for confirmation of the DD diagnosis and identification of Treponema phylotypes (PTs). RESULTS: High numbers of Treponema spp. were detected from all nine locations on the trimming equipment and chutes immediately after trimming, and T. phagedenis was detected on two or more locations in all but two herds, 1 and 19. There was a decline in the amount of Treponema spp. after washing and disinfection. The belly belt, the cuff, and the footrest on the chute had the highest proportion of positive samples after disinfection. The belly belt had the highest copy numbers of all nine locations (median = 7.9, max = 545.1). No Treponema spp. was detected on the hoof knives after disinfection. Treponema phagedenis, T. pedis, and Treponema phylotype 3 (T. refringens) were detected by FISH analysis of the biopsies. Treponema phagedenis was detected in biopsies from all herds except 1 and 19. CONCLUSION: This study shows that DD-associated Treponema spp. were present on the trimming equipment and chutes after trimming cows in DD-positive herds. Washing and disinfection reduced the load of Treponema spp. However, large differences in Treponema spp. between different locations were documented. High copy numbers on the grinder and the chute after disinfection, indicates that sufficient cleaning and disinfection of these locations is difficult, and that passive transfer of DD-associated treponemes (viable or not) is possible.

Digital dermatitis in Swedish dairy herds assessed by ELISA targeting Treponema phagedenis in bulk tank milk.

BACKGROUND: Digital dermatitis (DD) is a contagious hoof infection affecting cattle worldwide. The disease causes lameness and a reduction in animal welfare, which ultimately leads to major decreases in milk production in dairy cattle. The disease is most likely of polymicrobial origin with Treponema phagedenis and other Treponema spp. playing a key role; however, the etiology is not fully understood. Diagnosis of the disease is based on visual assessment of the feet by trained hoof-trimmers and veterinarians, as a more reliable diagnostic method is lacking. The aim of this study was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) on bulk tank milk samples testing for the presence of T. phagedenis antibodies as a proxy to assess herd prevalence of DD in Swedish dairy cattle herds. RESULTS: Bulk tank milk samples were collected in 2013 from 612 dairy herds spread across Sweden. A nationwide DD apparent prevalence of 11.9% (8.1-14.4% CI95%) was found, with the highest proportion of test-positive herds in the South Swedish regions (31.3%; 19.9-42.4% CI95%). CONCLUSIONS: This study reveals an underestimation of DD prevalence based on test results compared to hoof trimming data, highlighting the critical need for a reliable and accurate diagnostic method. Such a method is essential for disease monitoring and the development of effective control strategies. The novelty of ELISA-based diagnostic methods for DD, coupled with the disease's polymicrobial origin, suggests an avenue for improvement. Developing an expanded ELISA, incorporating antigens from various bacterial species implicated in the disease, could enhance diagnostic accuracy. The significance of this study is underscored by the extensive analysis of a substantial sample size (612). Notably, this investigation stands as the largest assessment to date, evaluating the application of ELISA on bulk tank milk for DD diagnosis at the herd level.

Field Study on the Prevalence of Ovine Footrot, Contagious Ovine Digital Dermatitis, and Their Associated Bacterial Species in Swedish Sheep Flocks.

Ovine footrot and contagious ovine digital dermatitis (CODD) cause lameness in sheep, affecting welfare and economics. Previous Swedish studies focused on individual slaughter lambs, leaving flock-wide prevalence less explored. This study examined the prevalence of footrot and CODD in Swedish sheep flocks, focusing on adult sheep. From 99 flocks, 297 swabs were analysed using real-time PCR for Dichelobacter nodosus, Fusobacterium necrophorum, and Treponema spp. Sampled feet were photographed and assessed using scoring systems for footrot and CODD. Results indicated footrot prevalences (footrot score ≥ 2) of 0.7% and 2.0% at the individual and flock levels, respectively, whereas there were no signs of CODD. The individual footrot prevalence was lower than that from a 2009 study but aligned with a 2020 study, both conducted on slaughter lambs. Dichelobacter nodosus, F. necrophorum, and Treponema spp. were found in 5.7%, 1.3%, and 65.0% of sheep, and in 9.1%, 3.0%, and 82.8% of flocks, respectively. Compared to the 2020 study, there was a notable decrease in F. necrophorum and Treponema spp., while D. nodosus was consistent. In conclusion, the findings show a low prevalence of footrot, CODD, D. nodosus, and F. necrophorum in Swedish sheep flocks. Continuous surveillance and owner education are important to maintain this favourable status.

Assessment of ATP-Bioluminescence and Dipslide Sampling to Determine the Efficacy of Slaughterhouse Cleaning and Disinfection Compared with Total Aerobic and Enterobacterales Counts.

Inadequate cleaning and disinfection (C&D) in slaughterhouses can cause bacterial contamination of meat, resulting in foodborne disease and reduced meat quality. Different methods for monitoring the efficacy of C&D procedures are available, but few studies have assessed their reliability. This study examined C&D efficacy in slaughterhouses and evaluated the diagnostic performance of methods for measuring surface hygiene. One red meat and one poultry slaughterhouse in Sweden were each visited on six occasions before and six occasions after C&D. Sampling points were sampled with: swabbing and plating for total aerobic bacteria (TAB) and Enterobacterales (EB); dipslides for total viable count; and ATP-bioluminescence tests. To evaluate the diagnostic performance of the dipslide and ATP-bioluminescence methods, the results were compared with (TAB) as a reference. In total, 626 samples were collected. For the majority of samples, TAB was lower after than before C&D and EB were mainly detected before C&D, indicating C&D efficacy. Greater reductions in mean TAB were observed in processing areas (2.2 and 2.8 log CFU/100 cm2 in red meat and poultry slaughterhouse, respectively) than in slaughter areas (1.3 log CFU/100 cm2 in both slaughterhouses). Approximately half of all samples were assessed as non acceptably clean (52% for red meat and 46% for poultry slaughterhouse) according to previously published thresholds. Critical food contact surfaces that were insufficiently cleaned and disinfected were plucking fingers, shackles, and a post-dehairing table. Cleaning and disinfection of drains and floors were inadequate. The ATP-bioluminescence method showed low specificity compared with the reference (TAB) in both the red meat (0.30) and poultry slaughterhouses (0.64). The sensitivity of dipslides was low (0.26) in the red meat slaughterhouse compared with TAB. A combination of ATP-bioluminescence and dipslides could provide more accurate estimates of C&D efficacy.

Novel Genotype of Streptococcus dysgalactiae subsp. equisimilis Associated with Mastitis in an Arabian Filly: Genomic Approaches and Phenotypic Properties.

Streptococcus dysgalactiae subsp. equisimilis (Sde) is a commensal bacterium of horses that causes opportunistic infections. The aim of the work was to study genotypic and phenotypic properties of the Sde strain related to equine neonatal mastitis. Sde was isolated from an 8 day-old filly and sequenced for genome analysis, antibiotic susceptibility tests and virulence factor (VF) assays. The Sde strain presented the novel emm-subtype stC839.12 and the novel multilocus-sequence type ST-670, which belonged to a specific equine genotype group. Although no specific genotypic mechanisms related to antibiotic resistance were found, it presented genes encoding efflux pumps and transporters pmrA, bmrC and lmrP. Genes encoding several putative VFs including emm, cpa, fbp-2, adcA, hyl, htrA, tig, slo, and ndk and loci-encoding phosphoenolpyruvate-protein phosphotransferase systems were identified. This is the first report of an equine neonatal mastitis case caused by a novel genotype and horse specific Sde strain.

Development of a multiplex quantitative PCR assay for simultaneous detection of Treponema phagedenis, Treponema pedis, Treponema medium, and 'Treponema vincentii' and evaluation on bovine digital dermatitis biopsies.

Bovine digital dermatitis (BDD) is a contagious foot disease with worldwide occurrence in dairy cattle. The disease causes lameness and reduced animal welfare as well as economic losses for the farmer. The aetiology is not fully established but associations have been made with Treponema spp. Today, BDD diagnosis is mainly based on visual inspection of cattle feet, therefore this study aimed to develop a multiplex quantitative PCR (qPCR) assay targeting Treponema phagedenis, Treponema pedis, Treponema medium, and 'Treponema vincentii' to aid in diagnosis. The assay was tested for specificity on 53 bacterial strains and in silico on 168 Treponema spp. genomes, representative of at least 24 species. In addition, 37 BDD biopsies were analysed and the results compared to another qPCR assay published during the study period, which we modified by combining into a multiplex qPCR. The qPCR developed herein had a detection limit of 10 copies of each target species per PCR reaction. Both qPCR assays showed 100% specificity when tested on bacterial strains, but the qPCR developed in this study detected 3.4% more T. phagedenis-positive biopsies of lesion category M1-M4.1 than the modified assay. To conclude, the developed qPCR assay detecting T. phagedenis, T. pedis, T. medium, and 'T. vincentii' has high analytical sensitivity and specificity and provides a useful complementary tool for diagnosis and epidemiological studies of BDD. The assay could possibly also be used for contagious ovine digital dermatitis (CODD) as similar bacteriological profiles have been suggested for BDD and CODD, especially regarding certain Treponema spp.

Survival of livestock-associated methicillin-resistant Staphylococcus aureus CC398 on different surface materials.

BACKGROUND: Zoonotic livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is widely spread in pig herds in many countries. However, the knowledge regarding the survival of LA-MRSA in the pig farm environment is currently limited. The aim of this study was to assess the survival of LA-MRSA on different surface materials found in the farm environment. The study investigated the survival of two different LA-MRSA strains belonging to the clonal complex (CC) 398 on four different surfaces: stainless steel, polypropylene plastic, K30 concrete and commercial concrete disk coupons. The survival of the bacteria over time was determined by the viable count method and, where possible, fitting a model to the observed data by using nonlinear least squares method to calculate the half-life ([Formula: see text]) for different strain and material combinations. RESULTS: The study showed that the half-life of the bacteria was longer on polypropylene plastic ([Formula: see text]=11.08-15.78 days) than on stainless steel ([Formula: see text]=2.45-7.83 days). On these materials, both LA-MRSA strains survived through the 14 week observation period. The bacterial decay was fastest on the concrete surfaces, where LA-MRSA became undetectable after 3-9 weeks. CONCLUSIONS: The survival of LA-MRSA in the pig farm environment may be affected by different surface materials. A more frequent sampling protocol (< 7 days) is needed to determine the half-life on concrete surfaces.

Antimicrobial Resistance in Vaginal Bacteria in Inseminated Mares.

Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of Escherichia coli to trimethoprim (p = 0.0006), chloramphenicol and (p = 0.012) tetracycline (p = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders.

Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.

Vaginal Bacteria in Mares and the Occurrence of Antimicrobial Resistance.

Antibiotics are added to semen extenders in insemination doses but their effect on the vaginal microbiota of the inseminated female is unknown. The objectives of this study were to define the equine vaginal microbiota and its antimicrobial resistance, and to determine whether it changes after exposure to antibiotics in semen extenders. Vaginal swabs were taken prior to sham-insemination (day 0), and again on days 3, 7, and 14 after insemination. Isolated bacteria were identified by MALDI-TOF and tested for antimicrobial susceptibility by microdilution. The bacteria isolated from the vagina differed according to reproductive status (brood mare or maiden mare), location (north or middle of Sweden), and the stage of the estrous cycle. Five bacterial species were frequently isolated from mares in both locations: Escherichia coli, Staphylococcus capitis, Streptococcus equisimilis, Streptococcus thoraltensis, and Streptococcus zooepidemicus. Overall, vaginal bacteria isolated from inseminated mares showed higher antibiotic resistance than from non-inseminated mares, suggesting a possible link between exposure to antibiotics in the semen extender and the appearance of antimicrobial resistance. The whole-genome sequencing of E. coli isolates from inseminated mares revealed some genes which are known to confer antimicrobial resistance; however, some instances of resistance in these isolates were not characteristic of induced AMR.

Prevalence of bacterial species associated with ovine footrot and contagious ovine digital dermatitis in Swedish slaughter lambs.

BACKGROUND: Ovine footrot and contagious ovine digital dermatitis (CODD) are contagious mixed bacterial infections with major impacts on animal health and production. In Sweden, ovine footrot and CODD were first detected in 2004 and 2019, respectively. In 2009, a voluntary control programme for footrot was established, and a prevalence study in slaughter lambs was conducted, however, the distribution of footrot and CODD-associated bacteria is still unknown. This study examined the prevalence of Dichelobacter nodosus, Fusobacterium necrophorum and Treponema spp., as well as the current prevalence of footrot and CODD, in Swedish slaughter lambs. RESULTS: A total of 2048 feet, from 512 slaughter lambs, were collected from eight slaughterhouses throughout Sweden in autumn 2020. All feet were visually examined for lesions of footrot and CODD and sampled for subsequent real-time polymerase chain reaction (PCR) analysis. Nine lambs (1.8%) had at least one foot affected with footrot (footrot score ≥ 2). A CODD grade 1 lesion was detected in a single lamb (0.2%). The prevalence of D. nodosus, F. necrophorum and Treponema spp. was 6.1%, 7.6% and 90.6%, respectively. The D. nodosus detected were benign strains. CONCLUSIONS: The prevalence of footrot in Swedish slaughter lambs has been significantly reduced, from 5.8 to 1.8%, during the past 11 years. This indicates that preventive measures, such as the national control programme and elimination of footrot from affected flocks, have been effective. A single lamb (0.2%) was found with a CODD lesion (grade 1). In Sweden, benign rather than virulent strains of D. nodosus seem to be the most common. Neither D. nodosus nor F. necrophorum were widespread among Swedish slaughter lambs, but both were more likely to be found in lambs with footrot. Treponema spp. was very commonly found in lambs with and without footrot, but there is a lack of information on the individual Treponema spp. present in Swedish slaughter lambs and their potential pathogenicity.

Differences in Genotype and Antimicrobial Resistance between Campylobacter spp. Isolated from Organic and Conventionally Produced Chickens in Sweden.

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. Susceptibility for tetracycline, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, and gentamycin was determined by microdilution. All 77 isolates from organic chickens were sensitive to all antibiotics, except two C. jejuni that were resistant to tetracycline. Of the 80 isolates from conventional chickens, 22.5% of C. jejuni and 11.1% of C. coli were resistant to quinolones and 5.6% of C. jejuni were resistant to tetracycline. Whole-genome sequencing resulted in 50 different sequence types of C. jejuni and six of C. coli. Nine sequence types were found in both organic and conventional chickens. Two of these (ST-19 and ST-257) included isolates from conventional broilers with different resistance phenotypes to the remaining isolates from conventional and organic broilers. There are management differences between the production systems, such as feed, breed, use of coccidiostats, and access to outdoor area. It is unlikely that quinolone resistance has arisen due to use of antimicrobials, since fluoroquinolones are not permitted in Swedish broiler production.

First report on outbreaks of contagious ovine digital dermatitis in Sweden.

BACKGROUND: Contagious ovine digital dermatitis (CODD) is considered widespread in the United Kingdom but was only recently reported in mainland Europe, as one outbreak in Germany. The disease can cause severe lameness in sheep and, if left untreated, can lead to total avulsion of the hoof capsule. CODD is considered to have multifactorial and polymicrobial aetiology, in which Treponema medium/Treponema vincentii phylogroup, Treponema phagedenis phylogroup and Treponema pedis are believed to play a significant role. Footrot and CODD have a close connection and footrot is considered an important risk factor for CODD. CASE: Lameness, mainly in lambs aged 1.5 months, was reported on a farm in Sweden in spring 2018. The animals showed no signs of footrot and the causative agent, Dichelobacter nodosus, was not found. CODD was suspected but not confirmed, and the clinical signs subsided when the animals were turned out to pasture. In February 2019, young lambs and ewes were lame again and this time CODD was diagnosed. After treatment, the whole flock was slaughtered later in 2019 due to CODD. In autumn 2020, CODD was diagnosed on another Swedish farm, this time as part of a mixed infection with D. nodosus. The animals were treated with footbaths in zinc sulphate 10% by the farmer, but lameness recurred soon afterwards. The animals were treated, but ultimately the whole flock was slaughtered. No connection was found between the two farms. CONCLUSION: The first two outbreaks of CODD in Sweden have been diagnosed and are described in this case report. If it spreads, CODD could have a negative impact on the Swedish sheep industry in terms of animal welfare, production and antibiotic use.

Identification of Transmission Routes of Campylobacter and On-Farm Measures to Reduce Campylobacter in Chicken.

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens.

Survival of Streptococcus equi subsp. equi in Normal Saline Versus Phosphate-Buffered Saline and at Two Different Temperatures.

Streptococcus equi subsp. equi causes strangles in horses. Sampling to detect carriers is important for the control of the disease, and maximizing the sensitivity of this procedure is necessary. To provide a basis for the choice of sampling solution and transport temperature for samples, comparisons were made between the survival of Streptococcus equi subsp. equi in normal saline versus phosphate-buffered saline and at two different temperatures (cold and room temperature). At present, normal saline is used to sample the nasopharynx as well as the guttural pouches, and the sampling solution is transported without special cooling. The results revealed no significant difference in bacterial concentration levels between the two sampling solutions, but a significantly higher concentration of viable bacteria in the samples kept cold compared with room temperature. Hence, a change of sampling solution is not warranted, but maintaining the cold chain during storage and transport to the laboratory may be important for clinical samples.

Potential transmission routes of Dichelobacter nodosus.

Footrot caused by Dichelobacter nodosus is a highly contagious bacterial disease affecting the claw of sheep and the main cause of lameness in these animals. It is not only an economic burden but also a serious animal welfare issue. More information about the transmission of D. nodosus is needed for effective footrot control programs. We therefore determined the prevalence of D. nodosus in sheep presented at shows and markets where commingling of animals occurs. Furthermore, possible transmission vectors during foot trimming were investigated and trimming knife decontamination protocols evaluated. Sheep at six markets and four shows were sampled and tested for the presence of D. nodosus by real-time PCR. Different vectors, such as trimming knives were tested by real-time PCR and for viable D. nodosus by culture. The prevalence of virulent D. nodosus in sheep presented at shows and markets ranged from 1.7% to 100%. Regions with an ongoing control program showed significantly lower prevalence. After trimming, positive real-time PCR and culture results were obtained from the knives, the hands of the claw trimmers as well as removed claw horn material whereas boots were only positive by real-time PCR. In conclusion, markets and shows pose a risk for transmission of D. nodosus. The risk of transmission is particularly high during claw trimming and recommended measures to limit this risk include wiping the knife with a disinfection towel, wearing and changing gloves after every sheep, as well as proper disposal of trimmed and infectious horn.

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus.

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

A distinct bacterial dysbiosis associated skin inflammation in ovine footrot.

Ovine footrot is a highly prevalent bacterial disease caused by Dichelobacter nodosus and characterised by the separation of the hoof horn from the underlying skin. The role of innate immune molecules and other bacterial communities in the development of footrot lesions remains unclear. This study shows a significant association between the high expression of IL1ß and high D. nodosus load in footrot samples. Investigation of the microbial population identified distinct bacterial populations in the different disease stages and also depending on the level of inflammation. Treponema (34%), Mycoplasma (29%) and Porphyromonas (15%) were the most abundant genera associated with high levels of inflammation in footrot. In contrast, Acinetobacter (25%), Corynebacteria (17%) and Flavobacterium (17%) were the most abundant genera associated with high levels of inflammation in healthy feet. This demonstrates for the first time there is a distinct microbial community associated with footrot and high cytokine expression.

Characterisation of Dichelobacter nodosus and detection of Fusobacterium necrophorum and Treponema spp. in sheep with different clinical manifestations of footrot.

The aim of this study was to determine the proportion of Dichelobacter nodosus, Fusobacterium necrophorum and Treponema spp. in sheep with different clinical manifestations of footrot compared to healthy sheep both at flock and individual level. The second aim was to characterise D. nodosus with respect to virulence, presence of intA gene and the serogroups. Swab samples (n=1000) from footrot-affected (n=10) and healthy flocks (n=10) were analysed for the presence of D. nodosus, F. necrophorum and Treponema spp. by real-time PCR and culturing (D. nodosus only). Dichelobacter nodosus isolates (n=78) and positive swabs (n=474) were analysed by real-time PCR for the aprV2/B2 and the intA genes and by PCR for the fimA gene (isolates only). D. nodosus was more commonly found in flocks affected with footrot than in clinically healthy flocks. A significant association was found between feet with severe footrot lesions and the aprV2 gene and between feet with moderate or no lesions and the aprB2 gene, respectively. F. necrophorum was more commonly found in flocks with footrot lesions than in flocks without lesions. No significant association was found between sheep flocks affected with footrot and findings of Treponema spp. or the intA gene. Benign D. nodosus of six different serogroups was detected in twelve flocks and virulent D. nodosus of serogroup G in one. In conclusion, D. nodosus and F. necrophorum were more commonly found in feet with footrot than in healthy feet. The majority of D. nodosus detected was benign, while virulent D. nodosus was only detected in a single flock.