“Non-healing” claw horn lesions in dairy cows: Clinical, histopathological and molecular biological characterization of four cases

The increasing prevalence of bovine digital dermatitis (BDD) contributes to a higher occurrence of secondary infections of exposed corium with Treponema spp. in bovine claws. “Non-healing” claw horn lesions (NHL) clinically resemble BDD lesions. They are severe, cause chronic lameness, and may persist for several months. They poorly respond to standard treatments of BDD and represent a serious welfare issue. In this study, four cases of NHL were classified clinically either as BDD-associated axial horn fissures (BDD-HFA; n = 3) or BDD-associated sole ulcer (BDD-SU; n = 1). In all four cases, pronounced multifocal keratinolysis of the stratum corneum, ulceration, and severe chronic lymphoplasmacytic perivascular to interstitial dermatitis were observed. All lesional samples tested positive for Treponema spp., Fusobacterium (F.) necrophorum, and Porphyromonas (P.) levii by PCRs. BDD-HFA lesions contained Treponema pedis as revealed by genetic identities of 93, 99, and 100%. Treponemes in the BDD-SU lesion were 94% homologous to Treponema phylotype PT3. Fluorescent in situ hybridization (FISH) revealed extensive epidermal infiltration by treponemes that made up > 90% of the total bacterial population in all four lesions. FISH also tested positive for P. levii and negative for F. necrophorum in all four cases, whilst only one BDD-HFA contained Dichelobacter nodosus. Our data point to BDD-associated treponemes and P. levii constituting potential etiological agents in the development of “non-healing” claw horn lesions in cattle.


Introduction
Bovine digital dermatitis (BDD) was first described by Cheli and Mortellaro in Italy in 1974 (1). Since then, the prevalence of the disease has continuously increased in many countries (2,3). The etiology of BDD is polymicrobial (4), with specific Treponema spp. being most commonly associated with disease development and progression (5)(6)(7). BDD typically involves the digital skin and is often classified using the M-stage scoring system (8). An increase in the prevalence of BDD also contributes to a higher occurrence of secondary infections of the exposed corium by Treponema spp. (9,10). Clinically, "nonhealing" claw horn lesions (NHL) are similar to BDD lesions and characterized by a moist, topical granular appearance and pungent fetid smell (11,12). In contrast to the classical forms of non-infectious claw horn diseases, Evans et al. (11) reported that NHL are typically more severe, cause chronic lameness, and poorly respond to state-of-art BDD treatment strategies. NHL may persist for many months, and thus, can seriously compromise the welfare of affected animals (13). NHL is also known as "BDD-associated claw horn lesion". Today, a curative treatment strategy for NHL is available (13)(14)(15). The latter consists of extensive debridement of loose horn and infected corium under local anesthesia, followed by topical application of copper sulfate, antibiotics (e.g., oxy-/ chlortetracycline), or salicylic acid under a bandage, and application of a block on the sound partner claw (14).
Molecular studies provide evidence of a strong association of three distinct phylogroups of BDD-associated Treponema spp. with NHL (11,16,17). The objective of the herein-reported study was to characterize four NHL cases in Swiss dairy cows with respect to their clinical appearance, histopathological features, and bacterial infection by a combination of histopathological and molecular biological techniques.

Case history
Two NHL-affected cows from the same farm were presented at the Clinic of Ruminants, Vetsuisse Faculty, University Zurich, one NHL case was diagnosed at the Clinic of Ruminants, Vetsuisse Faculty, University of Bern, and one NHL case was detected during a herd health visit at a dairy farm located in the canton of Fribourg, Switzerland.
All cows showed mild to moderate lameness for several weeks (locomotion score ranged between 2 and 3; Table 1) according to Sprecher et al. (19), with 1 = non-lame to 5 = severely lame. After applying a wooden block on the partner claw and cleansing of the affected area with 1%-povidone-iodine or Octenidine solution (Octenisan R Schülke und Mayr AG, Frauenfeld, Switzerland), all lesions were treated by extensive surgical debridement including the removal of loose horn and removal of granulation tissue under three-point local anesthesia using lidocaine hydrochloride (Lidocaine 2%, Streuli Tiergesundheit AG, Uznach, Switzerland). Thereafter, a wound dressing with diluted oxytetracycline (Engemycin R 10% injectable solution, MSD Animal Health GmbH, Luzern, Switzerland) or an octenidine ointment (Octenisept R Gel, Schülke und Mayr Ag, Frauenfeld, Switzerland) was administered topically onto all BDD-HFA, and bandages were applied. Treatment was repeated every 2-3 days until the wound healed, and then at weekly intervals until defects were covered by a sufficient and stable new horn. Management of the BDD-SU consisted of removal of granulation tissue under regional intravenous anesthesia using 20 mL of lidocaine hydrochloride, application of povidone-iodine ointment (Betadine R , Covetrus AG, Lyssach, Switzerland) under the bandage and weekly bandage changes until defects were covered by new horn.

Sample collection, processing, and DNA extraction
Affected feet were washed with 1%-povidone-iodine or octenidine solution. Subsequently, lesional tissue was obtained in the course of therapeutic debridement. Each tissue sample was longitudinally bidissected on a sterile petri dish using sterile forceps and #11 scalpel blades.
Sample processing for both molecular biological and histopathological analyses was done as described previously by Alsaaod et al. (20). For histopathological evaluation, one sample section was fixed in 10% neutral buffered formalin, embedded in paraffin, cut, and stained with hematoxylin and eosin or with the Warthin-Starry stain.
From the other sample section, DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. Purified DNA samples were stored at −20 • C until further use.

Histopathological evaluation
Histopathological evaluation was performed according to Read and Walker (21) modified by Klitgaard (18) to classify epidermal and dermal changes. Characteristic BDD-associated changes commonly include (i) a focally .

PCR assays and sequencing
DNA extracts were screened for the presence of treponemal DNA using consensus "total" Treponema (TT) primers 5 ′ /3 ′ (TT-PCR) designed by Moe et al. (22) according to an optimized protocol (23), which is described in detail by Alsaaod

Fluorescent in situ hybridization
For FISH analysis, serial 4-µm sections were prepared from formalin-fixed, paraffin-embedded biopsies and hybridized as described previously (25). In brief, hybridization was carried out at 45 • C for 16 h and a final probe concentration of 5 ng/µL. After hybridization, the slides were washed three times in pre-warmed (45 • C) hybridization buffer for 3 minutes and subsequently with washing buffer at the same time intervals. The sections were rinsed in water, air dried, and mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA) for fluorescence microscopy. The probe for domain bacteria was 5' labeled with fluorescein isothiocyanate (FITC) and all other bacteria probes were 5' labeled with the isothiocyanate derivative Cy3 (Eurofins MWG Operon, Ebersberg, Germany).

Clinical findings
BDD-HFA and BDD-SU lesions were severe, penetrating the horn capsule, and involving the corium. Lesions exhibited hypergranulation tissue covering the axial horn fissure (n = 3) or the plantar sole area (n = 1) and were classified according to the International Committee for Animal Recording Claw Health Atlas Kofler et al., (13) as BDD-associated axial horn fissures (BDD-HFA; Figure 1A), and BDD-associated sole ulcer (BDD-SU; Figure 1B), respectively. Figure 1 exemplarily depicts the gross pathological features of one case of BDD-HFA (A) and BDD-SU (B). The treatment duration ranged between 2 and 4 weeks until defects were covered by a sufficient and stable new horn.

Histopathological findings
All four lesions exhibited severe multifocal keratinolysis of the stratum corneum with ulceration (Figures 2A,B) as well as loss of the stratum granulosum. In three cases, the epidermis was acanthotic. Warthin-Starry stain pointed to the presence of spirochetes on the surface and within the epidermis in all samples, with spiral bacteria being observed (Table 1). All lesions also displayed severe, chronic lymphoplasmacytic perivascular to interstitial dermatitis.

PCR detection and sequencing results
All lesions tested positive by total Treponema PCR. Amplicon sequencing and subsequent BLAST alignment resulted in the identification of three T. pedis sequences from BDD-HFA lesions, showing 99% identity with T. pedis strain G2JD, 100% identity with T. pedis strain DD3F, and 93% identity with T. pedis strain G9JD, respectively. The amplicon detected from the BDD-SU lesion was 94% homologous to Treponema phylotype PT3 AM942447 (18). All lesions also tested positive for F. necrophorum and P. levii, whilst only one BDD-HFA scored positive for D. nodosus (Table 1). TT PCR from no-template, negative, and positive control yielded the expected results, thus confirming the authenticity of obtained results.

FISH results
FISH analysis revealed severe, extensive epidermal infiltration (score 3) by treponemes that made up 90% of the total bacterial population in all four cases ( Figure 3A). Additional FISH analysis of Treponema spp. positive biopsy samples revealed a mixed infection with T. phagedenis, T. pedis, and T. medium in one BDD-HFA, whereas another BDD-HFA was only infected by T. pedis ( Figure 3B). BDD-SU tissue tested FISH positive for the genus Treponema but was negative for all four Treponema phylotypes assessed. Moderate hybridization for D. nodosus was only noted in one case of BDD-HFA. All three BDD-HFA lesions exhibited a strong hybridization signal for P. levii ( Figure 3C). F. necrophorum could not be detected in any of the lesions (Table 1).

Discussion
Reports on the prevalence and biopathological features of bovine NHL have increased over the past years. Yet, many aspects of the etiopathogenesis of the disease remain unclear. To our knowledge, to date, NHL has not been histopathologically analyzed in combination with molecular biological techniques.
All four BDD-NHL-affected dairy cows originated from herds with a history of BDD and were kept in a freestall systems. Endemically BDD-affected herds are described to be at higher risk of developing NHL (9, 10, 26).
Severe multifocal keratinolysis of the stratum corneum, ulceration and severe, chronic lymphoplasmacytic perivascular to interstitial dermatitis in concert with spirochetal colonization were the predominant histological features noted in all four cases. These findings are consistent with the typical pathological features of BDD (18,27).
Treponema spp. have been reported as a cause of contagious ovine digital dermatitis (CODD) in sheep (28,29). In small ruminants and wildlife elk, treponemal infections typically lead to dermatitis along the sole, which can result in complete hoof avulsion in severe cases (29,30). More recently, BDDassociated Treponema spp. were detected in captive European bison (Bison bonasus) in Switzerland (31). In agreement with Evans et al. (11) and Sykora et al. (17), treponemal DNA was detected in all cases of NHL in our study. In the study by Sykora et al. (17), T. medium was almost exclusively found in BDD-WLD and BDD-SU, whereas T. pedis was equally detected in common BDD lesions and NHL. As a result, it was proposed that T. medium might play a key role in the pathogenesis of BDD-WLD and BDD-SU. In the present study, T. medium was only identified in one case Frontiers in Veterinary Science frontiersin.org . /fvets. . of BDD-HFA. Two of the four cases were positive for T. pedis as confirmed by FISH and PCR followed by amplicon sequencing. Therefore, further research is needed to clarify the role of different Treponema spp. by screening different types of NHL vs. common BDD lesions using Treponema spp.-specific detection methods. P. levii is a pleomorphic, gram-negative, anaerobic, rod-shaped bacterium possibly associated with bovine metritis (32), bovine necrotic vulvovaginitis (33), BDD (34) and bovine interdigital phlegmon (35). Whilst there is a lack of information regarding the presence of P. levii in bovine NHL, Staton et al. (36) identified P. endodontalis [which is highly associated with chronic oral infections in humans, (37)] by PCR in the vast majority of BDD-WLD and BDD-SU lesions. Thus, it was suggested that P. endodontalis could chiefly contribute to the development of NHL. Since P. levii was detected in three out of four NHL lesions as revealed by a strong hybridization signal, the assumption is supported that P. levii might indeed have a pathobiological role in BDD onset and progression. Further studies on higher numbers of NHL cases are needed to understand the role of P. levii in this and other types of claw diseases.
F. necrophorum was frequently found in BDD-WLD, BDD-SU, and BDD-associated toe necrosis in a previous study (36). These findings contrast with our results, as we did not detect F. necrophorum in the investigated NHL lesions. We hence question the proposed theory on a causative role of F. necrophorum in the pathogenesis of NHL. these bacteria in only 2/10 NHL lesions screened. While sheep are the primary hosts for benign and virulent strains of D. nodosus involved in the multifactorial pathogenesis of ovine footrot (38), cattle typically host benign D. nodosus strains (39). Therefore, a relevant role in the development of NHL seems unlikely.
One main advantage of FISH as a culture-independent molecular biological method compared to PCR techniques is that it can localize bacterial infection. Using FISH, a distinction can be made between superficial colonization and invasion of deeper epidermal layers, and well-established infection scoring methods can be used (18,25). Therefore, FISH constitutes a valuable tool to assess the involvement of different pathogens in the development of NHL, especially in combination with highly sensitive amplification techniques.
In conclusion, P. levii was detected in three of four cases of NHL, suggesting a potential synergistic activity of P. levii and BDD-associated treponemes in the development of bovine NHL. In contrast, present results do not allow a central role to be assigned to F. necrophorum or D. nodosus in the pathogenesis of NHL. The synergistic activity of P. levii and BDD-associated treponemes in the development of bovine NHL should be further investigated by the inclusion of a higher number of cases to show a consolidated association. Still, the results presented in this report provide new insights into the etiopathogenesis of NHL and encourage further research on this relevant claw disease.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary materials, further inquiries can be directed to the corresponding author.

Ethics statement
Ethical review and approval was not required for the animal study because lesional tissue was obtained in the course of therapeutic debridement. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions
MA was responsible for data collection, molecular analyses, and writing the first draft of the manuscript. ES and DD supported the data collection. CG performed the histological analyses. TJ and SB supported the data analyses. AS, SB, and JW edited the manuscript. AS supervised the study. All authors contributed to the manuscript and approved the final version.

Funding
This work was supported by the Heard Health Management Initiator Grant, Institute of Animal Pathology and Clinic for Ruminants (Vetsuisse Faculty, University of Bern, Switzerland).