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.

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.

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.

The occurrence of Treponema spp. in gingival plaque from dogs with varying degree of periodontal disease.

Periodontal disease is common in dogs and is initiated by gingival plaque composed of several hundred bacterial species. Some of these species have specifically been pointed out as potential periodontal pathogens, such as Treponema spp. Treponema spp. are difficult to culture and therefore the majority have been detected by culture-independent methods, such as PCR (Polymerase Chain Reaction). This leaves many Treponema spp. uncharacterized and unnamed. In this study, treponemes were investigated in gingival plaque from dogs with varying degree of periodontal disease with the aim to describe their occurrence and diversity in dogs. The methods used were culture, phase-contrast microscopy, PCR targeting the 16SrRNA-tRNAIle intergenic spacer region (ISR2), sequencing of the ISR2 and phylogenetic analysis. Treponema spp. were detected in samples from 10 out of 11 dogs and isolates were obtained from six dogs. Both healthy and periodontitis affected dogs were Treponema positive. Phylogenetic analysis, based on ISR2 sequences, revealed a large diversity of treponemes in the study population that were found to be distributed mainly in two groups, corresponding to the human oral treponeme phylogroups II (Treponema denticola) and IV (Treponema maltophilum) genetic groups. They were generally more distantly related to other treponemes in these groups. Treponemes from dogs with periodontitis and dogs with mild gingivitis without periodontitis did not differ in any obvious way. The results indicate that several phylotypes of oral treponemes are common in dogs regardless of periodontal status.

Detection of Treponema phagedenis-like antibodies in serum and bulk milk from cows with and without digital dermatitis.

Digital dermatitis (DD) in dairy cows is a widespread disease linked to infection with Treponema. The traditional diagnostic method is clinical inspection, which is subjective and laborious. We explored the performance of 4 different immunogenic proteins from Treponema phagedenis in a new antibody ELISA for analysis of serum or milk. Analysis of samples from 390 cows in 25 herds showed that the ELISA could distinguish the majority of cows with DD from healthy cows. By changing the cutoff and applying parallel or serial testing, high sensitivity or specificity could be achieved. The investigation indicated that aggregated test results can be useful in the assessment of a herd's DD status. In addition, analysis of bulk tank milk samples showed good agreement with results from individual cows. The test system could be useful in research on the epidemiology and immunology of DD.

Experimental inoculation of Treponema pedis T A4 failed to induce ear necrosis in pigs.

Ear necrosis is a syndrome affecting pigs shortly after weaning and is regarded as an animal welfare issue. The etiology is unknown but Treponema spp., predominantly Treponema pedis, are commonly detected in the lesions. Oral treponemes have been suggested as source of infection, transferred by biting and licking behavior. In this study, five pigs were intradermally inoculated with Treponema pedis strain T A4 with the aim of investigating if this strain would induce ear lesions. Three pigs served as controls. The inoculation was repeated after 29 days, and the study continued for 56 days. Serum samples were collected throughout the study and analyzed by ELISA for IgG antibodies towards T. pedis T A4 lysate. Skin biopsies were taken from the inoculation area at the end of the study. Gingival samples were collected and cultivated for treponemes, for comparison to the inoculation strain and to follow colonisation. The challenged pigs did not develop any clinical signs of infection and no spirochetes were detected in sections from skin biopsies. The number of Treponema-positive gingival samples increased during the study. In the challenge group, IgG towards the bacterial lysate peaked 7 days after each inoculation and decreased rapidly hereafter. In the control group a weak IgG response was observed after the second inoculation, possibly caused by the oral treponemes.

Genetic analysis of a Treponema phagedenis locus encoding antigenic lipoproteins with potential for antigenic variation.

Digital dermatitis (DD) is a painful and debilitating claw disease in cattle. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The occurrence of Treponema phagedenis in DD lesions, especially near the interface of healthy and diseased tissue, suggests that this species contributes to the development and/or progression of the lesions. In this study we characterized a genetic locus in T. phagedenis that contains coding regions for three antigenic proteins, PrrA, VpsA, and VpsB. Comparative analysis of homologous loci from fifteen strains suggests that prrA may be transposed into or out of this locus. Alterations in the copy number of TA repeats within the putative promoter region may regulate VpsA/B expression. The vpsA and prrA genes occur in allelic variants in different T. phagedenis isolates and may provide one explanation for the antigenic variation observed in T. phagedenis DD isolates.

Draft genome sequence of 'Treponema phagedenis' strain V1, isolated from bovine digital dermatitis.

'Treponema phagedenis' is considered to be a key agent in the pathogenesis of bovine digital dermatitis, an infectious foot condition of economic and animal welfare importance. We hereby report the draft sequence of 'T. phagedenis' strain V1. The draft genome assembly consists of 51 scaffolds comprising 3,129,551 bp and a GC-content of 39.9 %. Putative pathogenicity related factors have been identified in the genome that can be used in future studies to gain insight into the pathogenic mechanisms of 'T. phagedenis'.

Identification of immunogenic proteins in Treponema phagedenis-like strain V1 from digital dermatitis lesions by phage display.

Digital dermatitis (DD) is a contagious claw disease causing lameness in cattle, affecting both animal welfare and economics. In this study, shotgun phage display was used to identify immunogenic proteins in a strain (V1) of the Treponema phylotype closely related to Treponema phagedenis, indicated as a key agent in the pathogenesis of DD. A genomic phage library was constructed and selected against antibodies from a rabbit immunized with live strain V1 bacteria. A homolog to the immunogenic protein TmpA of Treponema pallidum subsp. pallidum was identified, as well as a putative phage tail tape measure protein (Ttm), and a putative proline-rich repeat lipoprotein (PrrA). The complete amino acid sequences of these proteins were predicted from a genomic sequence of strain V1 generated by 454 Sequencing™. The presence of these genes in ten Treponema spp. field isolates was investigated by PCR. The tmpA and ttm genes were detected in all T. phagedenis-like isolates while prrA was detected in four out of seven. None of the genes were detected in the three Treponema pedis isolates investigated. Recombinant proteins were produced and used in indirect ELISAs. For all three proteins, a majority of serum samples from cattle with DD (n=8) showed higher optical density values than samples from cattle without DD (n=7).

An IgG-binding protein A homolog in Staphylococcus hyicus.

Shotgun phage display was used to identify a homolog of the IgG-binding protein staphylococcal protein A in Staphylococcus hyicus type strain CCUG 15602/ATCC 11249. This bacterium is the causative agent of exudative epidermitis in pigs and can also cause mastitis in cattle. A protein with similar features as the originally identified protein A in Staphylococcus aureus was described; an YSIRK-type signal peptide, four IgG-binding domains, a putative peptidoglycan-binding domain, and a cell wall anchoring motif (LPXTG) was present. The highest degree of similarity was to a protein A homolog in Staphylococcus pseudintermedius. However, typical Xr polypeptide repeats present in the protein A of S. aureus and S. pseudintermedius could not be identified in the protein A of S. hyicus. The presence of the spa gene in ten porcine and eight bovine clinical isolates of S. hyicus was investigated by PCR. In all isolates, the spa gene could be detected but the amplicons were of two sizes. Sequence analysis of four selected PCR amplicons showed that only three IgG-binding domains were present in the protein A of clinical isolates generating a smaller spa fragment. The finding of spa in S. hyicus contributes to an increased understanding of potential virulence factors in this species.

Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938.

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known beta-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The beta-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

Sorting a Staphylococcus aureus phage display library against ex vivo biomaterial.

A phage display library made from Staphylococcus aureus DNA was sorted against a central venous catheter (CVC) that had been removed from a patient 2 days after insertion. After the first panning, approximately 50% of the clones encoded proteins known to interact with mammalian proteins. After the second and third pannings, fibrinogen-binding and beta2-glycoprotein I (beta2-GPI)-binding phage particles were clearly dominating. Proteins adsorbed to different CVCs were investigated using specific antibodies. Among the proteins probed for, fibrinogen was most abundant, but, interestingly, beta2-GPI was also detected on all tested CVCs.

Phage display reveals 52 novel extracellular and transmembrane proteins from Lactobacillus reuteri DSM 20016(T).

Extracellular and transmembrane proteins are important for the binding of bacteria to intestinal surfaces and for their interaction with the host. The aim of this study was to identify genes encoding extracellular and transmembrane proteins from the probiotic bacterium Lactobacillus reuteri by construction and screening of a phage display library. This library was constructed by insertion of randomly fragmented DNA from L. reuteri into the phagemid vector pG3DSS, which was previously developed for screening for extracellular proteins. After affinity selection of the library, the L. reuteri inserts were sequenced and analysed with bioinformatic tools. The screening resulted in the identification of 52 novel genes encoding extracellular and transmembrane proteins. These proteins were classified as: transport proteins; enzymes; sensor-regulator proteins; proteins involved in host/microbial interactions; conserved hypothetical proteins; and unconserved hypothetical proteins. Further characterization of the extracellular and transmembrane proteins identified should contribute to the understanding of the probiotic properties of L. reuteri.

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins.

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display.

A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.

Phage display as a novel screening method to identify extracellular proteins.

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.