Foot health and prevalence of Dichelobacter nodosus in 11 ungulate species at Berne Animal Park

suggests that captive wild even-toed ungulates do not pose a risk to the planned footrot control program.


Summary
Dichelobacter nodosus (D. nodosus) is the etiological agent of ovine footrot affecting mainly sheep worldwide, but also free-ranging wild ungulates such as Alpine ibex (Capra ibex ibex) and mufflon (Ovis orientalis orientalis).A nationwide ovine footrot eradication program is planned for the years to come, based on polymerase chain reaction (PCR)-testing of interdigital swab samples and regular footbathing.In this cross-sectional study, we clinically evaluated the foot health and analysed presence of D. nodosus in 11 different even-toed ungulate species (mainly European species) during a 13 months (2018-2019) period in Berne Animal Park.The foot lesions were scored for any clinical signs of pathologies as described in cattle and simultaneously for clinical signs of footrot as described for sheep, using a scale from 0 to 5 (while 0 describes clinically healthy feet and 5 loss of the horn capsule).From a total of 53 animals, 4-feet swab samples were taken from the interdigital cleft and subjected to real-time PCR assays to detect D. nodosus at animal level.Foot lesions were detected in five different species.In 3/5 muskoxen (Ovibos moschatus wardi), 7/12 Cretan wild goats (Capra hircus cretica) and 2/3 dwarf goats (Capra hircus aegagrus), they mainly consisted of white line disease, whereas in 9/10 European bison, dermatitis of the interdigital cleft was diagnosed.1/3 alpaca was diagnosed with chorioptic mange of the heel area.None of the examined animals showed clinical signs of footrot (score 0), and neither benign (aprB2-positive) nor virulent (aprV2-positive) D. nodosus were detected in any of the samples.This study provides additional information to facilitate an efficient ovine footrot control program in Switzerland and suggests that captive wild even-toed ungulates do not pose a risk to the planned footrot control program.In recent years, footrot diagnostics have been remarkably improved by establishing a competitive real-time polymerase chain reaction (PCR) method allowing detection and differentiation of the virulent and benign strains of D. nodosus directly from clinical samples. 17his, in turn, enabled the non-invasive swabbing of feet and early detection of animals harbouring D. nodosus with high sensitivity and specificity, and additionally to quantify the amount of bacteria present.
In Switzerland, the first footrot control program started in the 1990s in the canton of Grison (GR), more recently in the canton of Glarus (GL) and is currently still voluntarily in the other 24 cantons.The present footrot control program in sheep includes clinical examination and swab sampling from the interdigital cleft, followed by prudent removal of undermined horn without causing bleeding, and regular footbathing in disinfectant solutions of herds positive for virulent D. nodosus. 9However, information on footrot in animal parks and zoos is scarce, and there is a lack of knowledge concerning PCR diagnostics in diverse exotic ungulate species. 26For these reasons, we aimed to clinically evaluate the distal limbs of the diverse even-toed ungulate stock housed at Berne Animal Park and to estimate the prevalence of D. nodosus using the established real-time PCR.This study provides relevant additional information for the planned footrot control program in Switzerland in order to ameliorate the prospects of success.

Animal preparation
Random sampling of 53 even-toed ungulates from a total of 71 animals was performed during diagnostic, curative or prophylactic medical treatments as part of routine zoo veterinary practice, as well as during captures related to transports from the whole even-toed ungulate stock of Berne Animal Park (n = 11 different species and n = 71 animals totally kept).General anaesthesia for all species except Cretan wild goats (Capra

Introduction
Despite its clinical importance, epidemiologic information on foot health and diseases is scarce in the nondomestic animal literature. 26There are many predisposing factors that potentially contribute to foot disorders in captive nondomestic even-toed ungulates.Enclosure design, floor substrate, hygiene conditions and nutrition must meet with the natural history and welfare of the species to prevent health problems, including those of the feet. 8,22Spillover of foot pathogens between closely related species must be minimized by means of practical hygiene measures between enclosures and adequate quarantine investigations for new acquisitions.The inability to perform routine foot evaluations and trims in noncompliant species may further jeopardize foot health.
Dichelobacter nodosus (D. nodosus) is a gram-negative fastidious anaerobic bacterium and an essential pathogen involved in the multifactorial etiology of ovine footrot. 4,15The disease is reported worldwide as endemic, causing painful inflammation and lameness. 11,12The disease affects both domestic (mainly sheep) and to a much lesser extent free-ranging wild ungulates. 3,21A recent study in Switzerland estimated the true prevalence of virulent D. nodosus in sheep at 16.9% and 16.2% at animal and farm level, respectively. 2On the other hand, another recent study revealed a low prevalence of both benign (1.97%) and virulent D. nodosus strains (0.05%) in all four indigenous wild ruminants in Switzerland. 16Footrot is an important welfare issue and causes large economic losses throughout sheep flocks in many countries.In Switzerland, costs for disease management without control measures were estimated at CHF 172.3 million for the years 2014-2030. 25 nodosus isolates can be classified into benign and virulent types by their presence of the aprB2 or aprV2 gene which code for thermolabile or thermostable acidic proteases, respectively.Benign strains (aprB2-positive) tend to cause non-progressive inflammation of the interdigital skin (mild interdigital dermatitis), while virulent strains (aprV2-positive) are associated mainly with clinical footrot, interdigital ulceration and may progress to separation of the horn shoe from the underlying dermal tissue.17,18,24 Paarhufer in Zoologischen Einrichtungen kein Risiko für das geplante Eradikationsprogramm der Moderhinke in der Schweiz darstellen.

DNA extraction and real-time PCR
Individual swabs were placed immediately into an Eppendorf tube filled with 1ml of SV lysis buffer (4 M guanidine thiocyanate, 0.01 M Tris-HCl, 1% -mercaptoethanol) for DNA extraction according to an adapted protocol. 17The lysed samples were stored at +4 °C and transferred to the laboratory for DNA extraction within one week of sampling.DNA extraction was performed from a 500 µl lysate using a semi-automated extraction robot (KingFisher™ DuoPrime, Thermo Fisher Scientific, Reinach, Switzerland), and purified DNA was stored at -20 °C until further laboratory processing.Detection and typing of D. nodosus was performed using the competitive real-time PCR according to Stäuble et al. 17 including an internal positive extraction and PCR inhibition control. 13This real-time PCR distinguishes between the presence of the gene aprV2 encoding the thermostable protease AprV2, and the gene aprB2 coding for the thermosensitive protease AprB2 of D. nodosus in sheep.All real-time PCR reactions were analyzed in duplicate, with a mean cycle (Ct) threshold of <40 rated positive.

Ethical statement
The study protocol was approved by the animal experimentation committee of the canton of Bern, Switzerland (permission # BE89/18, 30672).

Results
Overall, 53 animals of a total of 71 individuals from 11 different even-toed ungulate species kept at the Berne Animal Park were sampled between November 2018 and December 2019.Involved species, demographic data and the results of the clinical findings are presented in Table 1.In 3/5 muskoxen (Ovibos moschatus wardi), 7/12 Cretan wild goats and 2/3 dwarf goats, foot lesions were observed, mainly exhibiting signs of white line disease (WLD), apart from single cases of horn fissures and scissor claws.Feet of both fore and hind limbs were affected, and lesions were observed in the abaxial as well as the axial aspect of the claws.In 9/10 European bison (Bison bonasus), signs of active dermatitis of the interdigital cleft were diagnosed, which affected both the fore and hind limbs.one of three alpacas (Vicugna pacos) presented with scaly lesions of the heel area of all four feet and skin scrapings revealed Chorioptes sp. as the etiologic ectoparasite.None of the examined animals showed any clinical signs of footrot (score 0).The results of the competitive real-time PCR revealed the complete absence of both benign and virulent strains of D. nodosus (Table 1).mouse-deer (Tragulus javanicus), a mixed syringe of medetomidine (Medetomidin HCL, Christoffel Apotheke, Bern, Switzerland or Dorbene, Graeub, Switzerland (0.04-0.08 mg/kg) and ketamine (Ketamin HCL, Christoffel Apotheke, Bern, Switzerland or Ketasol-100, Graeub, Switzerland, 1.5-3.0mg/kg) was applied per estimated body weight (bw) via blow pipe or dart gun (Daninject, Gelsenkirchen, Germany) in the shoulder or thigh musculature.The animals were placed in right lateral recumbency, received 0.1 l/min oxygen per 10 kg bw via nasal probe and vitamin E and selenium.For reversal, atipamezole (Alzane, Graeub, Switzerland, 0.2-0.4mg/kg) was administered at the end of the procedure.In total, 32 medetomidine-ketamine anaesthesias were performed.Minor complications occurred in a single muskox (superficial plane of anaesthesia with spontaneous movements at the end of the procedure) and two alpacas (transient respiratory depression and apnoea, treated with intubation and assisted ventilation), and recovery was uneventful in all the animals.Javan mouse-deer were trapped in an airtight box and anaesthesia was induced by 5.0 vol.% of isoflurane (Isoflo, Zoetis, Switzerland) in 2.0 liter oxygen, followed by maintenance with 2.0 vol.% of isoflurane in 0.5 l/min oxygen administered per face mask.

Collection of swab samples and foot lesion description
Samples were collected between November 2018 and December 2019.All four feet were examined for clinical signs of footrot and scored according to the BGK (Swiss Health Service for Small Ruminants) and adapted from Egerton and Roberts 6 using a scale from 0 to 5 (while 0 describes clinically healthy feet and 5 loss of the horn capsule).After removing the dirt with a dry paper towel, the feet were sampled with sterile, dry cotton swabs (2 mm × 15 cm, Stericlin, Germany) by rubbing the swab in the interdigital cleft of all four feet of the respective animal using one clean quarter of the same swab for each foot according to a standardized protocol. 14Attention was paid that the swabs had direct contact to the skin.Thereafter, standardized photographs were taken of all feet with dorsal and plantar views, digitally stored and blinded at animal level.Using these photographs, foot lesions were finally scored by the two trained and experienced first and last study authors according to the International Committee for Animal Recording (ICAR) claw health atlas (ICAR, 2015) for cattle (https://www.icar.org/Documents/ICAR_Claw_Health_Atlas.pdf,accessed 25

Discussion
We assessed for the first time the foot health and the prevalence of D. nodosus in 11 different even-toed ungulate species from Berne Animal Park.The sample size allowed a reliable estimate of the foot health status of the stock, since more than two thirds of the individuals were sampled.The comparison of our findings with the currently reported data in other wild and domestic ungulate species from Switzerland is important to understand the dynamics of footrot and its interaction between domestic and wildlife species. 16Further investigations are required to evaluate the true prevalence of D. nodosus in Swiss animal parks and zoos including many other exotic even-toed ungulate species.
The presence of benign and virulent strains of D. nodosus was found to be associated with severe foot lesions in Alpine ibex (Capra ibex ibex). 3,16,23A recent study in Switzerland reported the presence of both benign and virulent strains of D. nodosus in free-ranging Alpine ibex associated with severe lesions consist-ent with typical footrot in sheep. 16Despite the presence of both D. nodosus strain types in two clinically affected Alpine ibex, the authors reported that the infection with D. nodosus in Swiss free-ranging wild ungulates is only sporadic and thus maintenance and spread of this pathogen is unlikely.In accordance with these findings, in our study none of the examined species, involving three Swiss, five European, two domestic and a single exotic wild ungulate species, showed any clinical signs of footrot (all scored as 0).The absence of clinical footrot was further confirmed by the results of the competitive real-time PCR that showed the absence of both benign (aprB2-positive) and virulent (aprV2-positive) strains of D. nodosus.However, to prove a continuous footrot-free status, the biosecurity measures already in place and regular sampling and testing need to be continued.As a biosecurity measure, every even-toed ungulate that is introduced to Berne Animal Park passes through a standardized quarantine, and in this period, the feet are examined for possible foot pathogens.Sheep are primary hosts for both virulent and benign D. nodosus, while cattle carry benign D. nodosus. 2 Previous studies showed a high prevalence (up to 88%) of benign D. nodosus, but absence of virulent D. nodosus in the Swiss cattle population, 1,2 which seems epidemiologically irrelevant considering the fact that the national footrot eradication program of sheep focuses on the elimination of virulent strains (aprV2-positive) only.However, special attention is warranted when keeping sheep with other potentially susceptible ungulate species such as ibex and mufflon (Ovis orientalis orientalis).
Foot lesions were detected in 5/11 species and mainly consisted of WLD, apart from single cases of horn fissures and scissor claws.In cattle, these conditions are known as non-infectious foot lesions that affect the claw horn by multifactorial causes such as metabolic changes around calving associated with weakening of the connective tissue of the hoof suspensory structures, low body condition, poor claw conformation, poor flooring conditions, low digital cushion thickness and prolonged intervals of foot trimming. 5,7,10,19,20Based on the results of this study, regular foot trimming has been established for Cretan wild goats, dwarf goats and muskox.
In 9/10 European bison, dermatitis of the interdigital cleft of unknown origin was diagnosed, and research is currently in progress to determine the etiology of these lesions.

Table 1 :
Clinical foot health findings and the prevalence of Dichelobacter nodosus (D. nodosus) in 53 even-toed ungulates from Berne Animal Park, sampled between November 2018 and December 2019.