Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

Background

Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further.

Methods

Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline.

Results

Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern.

Conclusion

This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.     

Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle

Digital dermatitis (DD) is an infectious claw disease of cattle that causes painful lesions, principally along the coronary band of the claws. In the US alone, the estimated economic impact of DD is estimated to be $190 million. The etiology of DD remains unclear and there is no reliable laboratory test, so DD is most often diagnosed clinically. Spirochetal bacteria of the genera Treponema have been implicated in DD infections following their isolation using culture techniques, serological detection of bovine antibodies against treponemes, and amplification of treponemal 16s DNA sequences by PCR. During in vitro growth of spirochetes and treponemes isolated from DD, morphological changes have been observed indicating the presence of a spiral form and an encysted form. It is not known why encysted forms appear or what role they have in the progression of DD. The current study established growth curves for three subtypes of treponemes, Treponema denticola-like, Treponema phagedenis-like, and Treponema medium-like, while photographically monitoring changes in morphology. In addition to observing spiral and encysted forms, two intermediate forms were also observed. These appeared as either spiral forms with spherical bodies or as enveloped clusters of granules. The observation of encysted forms adds further support to the theory that treponemes causing recurrent infections deep in bovine skin have mechanisms to facilitate persistence and the chronic character of DD.     

Detection of spirochetes by polymerase chain reaction and its relation to the course of digital dermatitis after local antibiotic treatment in dairy cattle

The aim of the study was to monitor the course of digital dermatitis after local antibiotic treatment in an experimental group (treated on diagnosis) and a control group (treated 5 days later). The present study was carried out on 2 farms involving 18 animals. Monitoring was performed by means of clinical findings and detection of spirochetes on the surface of the lesions, using a polymerase chain reaction. Superficial wound smears were taken before and after treatment. Twelve animals on both farms followed the classical healing process, but six animals responded poorly to treatment. We observed that without treatment, there was no self-cure in the control group within 5 days. There was a significant improvement in the clinical condition of all animals after treatment on both farms, during the follow-up period. The time until reappearance of new digital dermatitis lesions was not significantly different between the experimental and control group, but it was different between the two farms which could be due to the influence of farm factors. Using primers specific for Treponema denticola and Treponema vincentii, all the disease stages had at least one positive polymerase chain reaction result indicating the presence of spirochetes in samples of all the disease stages during the healing process. This implies that the spirochetes are not completely eradicated from the surface of the lesions after treatment. It was also observed that the classical ulcerative disease stage (M2) had relatively more positive polymerase chain reaction results compared to any other disease stage, showing a possible link between the presence of spirochetes and clinical disease.     

Survey for papillomatous digital dermatitis in Australian dairy cattle

OBJECTIVE: To determine whether Treponema-associated papillomatous digital dermatitis (PDD) occurs in Australian dairy cattle. DESIGN: Mail-out questionnaire and histological and bacteriological examination of biopsy tissue from suspect PDD lesions. PROCEDURE: The questionnaire was mailed to 375 veterinarians to evaluate their knowledge of PDD, determine if they had observed the disease in Australian dairy cattle, and to request biopsy material from suspicious cases. Biopsies were examined for histological and bacteriological evidence of PDD, including for the presence of spirochaetes. RESULTS: Eighty-eight replies to the questionnaire were received (23.5%). Of 52 respondents who were aware of PDD as a possible cause of lameness, 26 reported observing the condition in Australian cattle. Of 32 respondents who were unaware of the condition, 6 reported observing lesions that might have been PDD. The majority of reports of PDD-like lesions came from the southern Australian states, the condition occurring during periods of high rainfall and proving responsive to topical or parenteral application of antimicrobials. Biopsies from five erosive lesions showed histological similarity to PDD whereas biopsies from five proliferative lesions were consistent with chronic inflammation, fibroma or cutaneous papilloma. The presence of spirochaetes was not demonstrated in any of the lesions by histological or bacteriological methods. CONCLUSION: Anecdotal reports and analysis of biopsy material confirm that a condition similar to PDD does occur sporadically in dairy cattle in southern Australia. However, this condition has so far not been shown to be associated with the presence of spirochaetes in the lesions.     

Treatment strategies for digital dermatitis for the UK

Digital dermatitis is a worldwide disease causing lameness in cattle, especially when housed. Despite its widespread nature there are relatively few peer-reviewed articles providing experimental data concerning its treatment and control. At the risk of descending to anecdote, this review attempts to summarise the current state of knowledge. Three approaches to digital dermatitis treatment have been used: (1) systemic antibiotics, (2) individual topical treatment, and (3) mass topical therapy using a footbath. Systemic antibiotics have become a rare treatment because of their cost and perceived lack of efficacy. Individual topical treatment, mainly with antibiotic preparations, is the most commonly reported method. It is widely considered the treatment of choice by virtue of reliable information about efficacy, the advantage of precise targeting of antibiotic usage and the requirements of legislation. However, at least in the UK, mass treatment of digital dermatitis by foot bathing is commonly used on many farms. In some cases, for example with antibiotic footbath solutions, these treatments create legislative, health and safety and environmental difficulties. Despite the widespread use of many different treatments, there is a lack of good comparative data and the consequence is a very variable and somewhat anecdotal approach in the field. There is a pressing need for well-researched treatment strategies for different herd situations.     

Antibodies to Borrelia spirochetes in sera from Swedish cattle and sheep

In the southern parts of Sweden a Borrelia infection transmitted by the tick Ixodes ricinus may affect man, In the present study antibodies to Borrelia spirochetes were studied in sera from 58 cows, 68 calves and 13 lambs from areas in southern Sweden where Ixodes ricinus occurs. For comparison, serologic studies were also performed on 88 cows and 10 lambs from the northern parts of Sweden.Serum titers of > 80 were found in 14 of the calves and 23 of the cows from southern Sweden but in only 1 of the cows from northern Sweden. In 11 of the lambs from the south a serum titer of > 40 developed. None of the lambs from the north had a serum titer of > 40. The results indicate that cattle and sheep in certain areas of Sweden are exposed to Ixodes ricinus-borne Borrelia spirochetes.In 9 of the lambs from southern Sweden: an endemically occurring arthritis had developed. The possibility that this arthritis may be caused by Borrelia spirochetes is discussed.     

Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows

Digital dermatitis (DD) of cattle leads to lameness and a decrease of milk production and is responsible for major economic losses worldwide. Although a bacterial aetiology is generally accepted, it still is unclear which microorganisms cause and/or maintain the disease. Recently, a previously undiscovered bacterial species, Guggenheimella bovis, has been isolated from the front of two DD lesions in Swiss cattle and suggested as a potential pathogen. The aims of the present study were to determine the prevalence of G. bovis in 58 German cows suffering from DD via dot blot hybridization, and to analyse the spatial distribution of G. bovis within the affected tissue by fluorescence in situ hybridization (FISH). A species-specific probe, GUBO1, was designed and evaluated. In none of the 58 samples Guggenheimella could be detected, while cultured G. bovis was reliably identified by GUBO1. Further FISH experiments were carried out on two additional biopsies of Swiss cattle tested positive for G. bovis by quantitative PCR and permitted visualization of the newly discovered bacteria in situ. In these biopsies G. bovis proved to be tissue invasive forming characteristic spherical microcolonies not only within the bacterial biofilm but also in seemingly unaffected parts of the tissue not yet reached by the advancing bacterial front. Although the presence of G. bovis does not constitute an essential premise for DD, it seems likely that the bacterial species involved in DD vary, and that in some cases G. bovis is crucial for the development of DD lesions.     

Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle

A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes. An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting. Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B. burgdorferi and the treponemes. Significant correlations were shown between antibodies to B. burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes. In Western blotting of B. burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands. For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa. Polyclonal antibodies to Treponema denticola and T. vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen. Monoclonal antibodies to B. burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting. The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis.     

A clinical trial to assess the use of sodium hypochlorite and oxytetracycline on the healing of digital dermatitis lesions in cattle

The increased frequency of diseases, especially those of the hoof, cause economic losses, such as premature culling of affected animals, decreased milk production, weight loss, reduced fertility, and the high costs of treatment. A great variety of hoof conditions may affect cattle, one of them is digital dermatitis. These conditions are probably due to multiple factorial diseases and present with similar clinical signs. Bovine lameness is typically treated by foot trimming and debridment of the lesions, coupled when necessary with systemic antibiotics and therapeutic footbaths, which results in a clinical cure in the majority of the cases. The objective of this study was to evaluate the topical action of sodium hypochlorite associated with the systemic use of oxytetracycline for the treatment of wounds clinically diagnosed as bovine digital dermatitis. One hundred and twenty Holstein cattle varying ages from 1 to 9 y and presenting the clinical signs of digital dermatitis, were used in this study. Group 1 (G1) received topical treatment with a 1% sodium hypochlorite footbath twice a day for 30 d and 4 treatments of parenteral oxytetracycline (10 mg/kg bodyweight, IM, q48h). Group 2 (G2) received only the topical treatment with 1% sodium hypochlorite, as described for G1. Group 3 (G3) received only with parenteral oxytetracycline, as described for G1. Group 4 (G4) was treated exclusively with a dicloro divynil pirrolidona, ortoiododimetil, para-nitofenil-fosforotioato in a vegetal tar-based ointment, immediately after the surgery. After 45 d, the recovery rates were as follows: G1, 86.67%; G2, 73.33%; G3, 56.67%; and G4, 50%. The surgical treatment of digital dermatitis with subsequent treatment with oxytetracycline systemically and 1% sodium hypochlorite topically was the most effective for the convalescence of cattle bearing wounds similar to digital dermatitis.     

Characterisation of a type 2 bovine viral diarrhoea virus isolated from cattle in the UK

Two genotypes of bovine viral diarrhoea virus (BVDV) are recognised. Type 2 was first recognised when virulent strains caused significant losses among cattle in North America. Subsequently, BVDV type 2 has been found in many other countries, but recent studies have shown that only type 1 BVDV is circulating in the UK herds (sheep and cattle) with type 1a predominating. During routine genotyping of UK BVDV isolates, a type 2 isolate was identified. Phylogenetic analysis of the 5'-untranslated region of the viral genome showed it to be a BVDV type 2a, most similar to a low virulent US strain of BVDV type 2. Antigenic typing with a panel of monoclonal antibodies verified this classification. This is the first confirmed isolation of BVDV type 2 found circulating in the UK.     

Prevalence of digital dermatitis in first lactation cows assessed at breeding cattle auctions

The prevalence of digital dermatitis (DD) in first lactation cows (FLCs) presented at 10 breeding cattle auctions was determined in a single auction centre in Austria over a period of 14 months. At each auction, FLCs were selected randomly for claw examination from the monthly auction catalogue. After obtaining owner consent, the hindclaws were examined in a walk-in crush. The type of lesion (M1–M4) and the diameter of DD lesions together with claw horn lesions and the trimming status of the claws were evaluated.From a total of 1110 FLCs registered for the ten auctions, 399 (36%) were randomly chosen for examination and 199 FLCs (17.9%) could be defined. In 63 cows (15.7%), the owners refused consent. A total of 24 FLCs were found to have DD lesions on one or both hindlimbs, resulting in a DD prevalence of 12.1%. The size of lesions ranged from 0.5–3 cm in diameter. No influence of environmental factors (such as season or housing) on the prevalence of DD could be found. In 30/199 (15.1%) of FLCs examined, claw horn lesions such as double sole, white line disease and sole ulcer were observed, but both claw horn and DD lesions were seen in only three cows. Potential buyers at auctions should be aware of both conditions, as the 12.1% prevalence of DD and the 15.1% prevalence of claw horn lesions in FLCs are considerable. Critical evaluation of claw health and DD is essential when purchasing breeding cattle.     

Some relationships between spirochaete infections and digital dermatitis in four UK dairy herds

Digital dermatitis, presenting as a focal necrotic dermatitis on the skin of cows' feet adjacent to the interdigital space, has been associated with dermal infection by spirochaetes. Lesions cause severe, transient lameness. An enzyme-linked immunoassay (ELISA) was used to measure IgG(2) antibody titres against three different spirochaetes: USA bovine Treponema isolates 1-9185MED and 2-1498, and a UK strain of Borrelia burgdorferi B31. Logistic models were used to describe lesion prevalence in terms of the antibody titres, herd and milking status, year, lactation number, and yield. The antibody titres and milking status (P<0.001) dominated the logistic fits, with the other variables adding little. Generalised linear models were used to describe the antibody titres in terms of the presence or absence of lesions, herd, year, lactation number, and yield. The presence or absence of lesions had the most significant effect for all three antibodies (P<0.001). Neither the herd effects nor the effects due to lactation number or yield showed consistent patterns.     

Prevalence of papillomatous digital dermatitis among culled adult cattle in the southeastern United States

OBJECTIVE: To determine prevalence of papillomatous digital dermatitis (PDD) among culled adult dairy and beef cattle in the southeastern United States. ANIMALS: 815 cattle examined during 4 visits to a slaughterhouse. PROCEDURE: The left hind foot of each animal was examined for gross lesions of PDD. Breed and sex of the animals were recorded. Lesions were examined histologically for pathologic changes and bacteria, especially spirochetes. RESULTS: 22 of 76 (29%) dairy cattle and 29 of 739 (4%) beef cattle had gross lesions of PDD. Detection of lesions was not associated with sex of dairy cattle, but male beef cattle were more likely to have lesions of PDD than were female beef cattle. Histologically, acute and chronic lesions were seen; the most severe changes were localized to the stratum corneum. Spirochetes were seen in lesions from 31 of 51 (61%) cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that PDD is common among culled adult cattle. Prevalence was higher in culled adult dairy cattle than in culled adult beef cattle.