ABSTRACT

Digital dermatitis is a highly prevalent painful lesion affecting the feet in dairy cattle. Even though the pathogenesis has been subject of investigation since 1974, there is still a lack of knowledge about the spread of the disease among cows within a herd as well as between herds. The purpose of this study was to monitor transmission of digital dermatitis under experimental conditions between naïve heifers and affected animals, to monitor the changes in clinical appearance, microbial colonisation of the skin as lesions progressed and to apply a q-PCR for the detection of Treponema spp. in faecal samples. Eight heifers with clinical normal digital skin were housed with 5 heifers with severe digital dermatitis lesion for 8 weeks on a solid concrete floor with an accumulating layer of slurry. Digital skin was examined daily and lesions were clinically scored. Skin biopsies were taken from the healthy heifers at introduction and weekly from all lesions for histopathological evaluation and fluorescence in situ hybridization. None of the healthy heifers developed digital dermatitis and in 4 out of 5 infected heifers the lesions healed during the study. All samples from healthy skin were negative for Treponema spp. and one sample were positive for Dichelobacter nodosus. Colonization of healthy skin could not be identified in this study. There was no significant relation between clinical scoring of the lesions and histopathological score and the presence of Treponema spp. There were however a significant relation between the prevalence of Treponema spp. in the skin and severity of changes in epidermis and dermis. By qPCR all the healthy heifers were found to excrete Treponema spp. in their faeces.

1. Introduction

Digital dermatitis (DD) is the most common cause of lameness in Danish dairy herds. A study performed in 2003 showed that 89% of Danish Holstein dairies have cows infected with DD and the mean herd level prevalence was 21% [1].

The pathogenesis of DD has been intensely investigated during the last decades. DD appears to be polymicrobial, since a wide variety of bacteria have been isolated from lesions [2,3]. Recently laboratory techniques, has made the identification of bacteria such as spirochetes possible within the epidermis and dermis using cultureindependent methods. More than 16 different Treponema phylotypes have been identified in DD lesions. The most commonly found bacteria in association with DD are treponemes belonging to three different clusters T. phagedenis-like [4-7], T. refringens-like [8,9] and T. denticola-like [10]. Even though the microbiological background for DD has been intensely investigated; there is still uncertainty about the spread of the disease. The ability of the causative agents to survive in the environment is unknown and therefore the knowledge of contamination within a herd is yet to be elucidated. DD has been called contagious and infectious [11], however, experimental transmission is difficult [12]. Only two times has an experimental spread of DD been reported as successful. Both studies had to macerate the skin and have anaerobic conditions unlike conditions seen with natural infection [13,14].

It is obvious from clinical studies and reports of outbreaks that DD do spread among cows in a herd; however the routes of disease spreading have not been elucidated. The development of the lesion and colonization of the pathogens in the skin prior to clinical DD have not been investigated, which means that the initial causative agents have not been identified. Studies have examined healthy skin in the perimeter of lesions and have not been able to show the presence of Treponema spp. [8]. Several studies have described finding Treponema spp. in the GI tract of bovine [15-18] and these treponemes are probably shed in feces, however the treponemes found associated with digital dermatitis have not been isolated from GI samples or feces [15]. The reservoir for DD Treponemes remains uncertain.

Naïve young stock/young heifers have previously been described to be especially susceptible to DD [19-21] and moisture and detrimental effect of urine and faeces are known risk factors for DD [21,22].

The purpose of this study was 1) to monitor transmission of digital dermatitis under experimental conditions between naïve heifers and affected animals; 2) to evaluate the relation between clinical macroscopic appearance (lesion stage) of DD and histopathological changes and microbial colonization of the skin and 3) to apply a qPCR for the detection of Treponema spp. in faecal samples.

2. Materials and Methods

2.1. Animals

Two maiden Holstein heifers 13 months of age from a DD-free tie stall herd (herd 1) and six maiden Holstein heifers 15 months of age from a DD-free free stall herd (herd 2) were housed together in a stall with solid concrete floor that measured 14 m by 8 m. Twelve hours a day the heifers has access to an additional area covered with deep straw bedding measuring 6 m by 8 m. During a period of four weeks the eight heifers build up a layer of approximately 40 cm of slurry on the concrete floor. After the four week introduction-period one first lactation Holstein cow (herd 3) with clinical lameness score 4/5 [23] and DD on both hind legs, from an endemic infected free stall herd, were introduced to the eight heifers. Following another four week period four maiden Holstein heifers (herd 4) from an endemic infected free stall herd were introduced to the group. All heifers had severe DD in the hind legs and a lameness score of 3/5 or 4/5 [23].

2.2. Examination

Before introduction to the experimental housing facility the heifers were examined in a trimming chute. Photographs were taken of all four limbs, the claws were trimmed and all lesions were recorded. Punch biopsies (6 mm, Kruuse, Denmark) were taken in the digital skin proximal of the heel horn on all hind legs following local anaesthesia of the skin, with 10 ml of Lidocain (20 mg/ml). No treatment or bandage was applied following biopsy sampling.

After introduction to the group all animals were examined on a daily basis. The examination included evaluation of general appearance, temperature, heart rate, respiratory rate, rumen contractions, faeces consistency, hydration, and they received a lameness score and were palpated for soreness of the digital skin. Once a week the heifers were examined in the trimming chute. The examination in the trimming chute included inspection and palpation of the digital skin. Faeces were removed with a gloved hand simultaneously with the palpation, leaving the skin visible but not clean, skin/lesions were scored for lesion stage and biopsies were sampled from all lesions.

2.3. Classification of DD Lesion Stage

DD was recorded using a standardised scoring system comprising five stages (M0-M4) [24]. If different stages of DD were present, the lesion was scored according to the most predominant stage of DD. M0: Normal skin, M1: Early stage of DD with a circumscribed granulomatous area, 0 to 4 cm in diameter, which lies at the epithelial surface or up to 2 mm underneath it. M1 is generally not painful on palpation. M2: Classical ulcerative stage of DD, with granulomatous tissue. M2 is often painful on palpation. M3: Classical ulceration of DD in the process of healing covered by a scab. M3 is generally not painful on palpation. M4: The chronic stage of DD are dyskeratotic or hyperkeratotic and can present themselves with a proliferative aspect. M4 is generally not painful on palpation.

2.4. Biopsies

Biopsies were fixed in 10% neutral buffed formalin, dehydrated and embedded in paraffin blocks. Serial tissue sections (4 µm) were cut and mounted on Super Frost + slides (Menzel-Gläser, Germany) for haematoxylin and eosin (H&E) stain and fluorescence in situ hybridization (FISH).

2.5. Histopathological Evaluation

All samples were evaluated histopathologically in H&E stained sections. Epidermal changes were scored 0 to 4 (0 = normal epidermis, 1 = hyperkeratosis and epidermal hyperplasia, 2 = focal, 3 = moderate, and 4 = severe acanthotic epidermis with or without parakeratotic papillomatous proliferation and ballooning degeneration of keratinocytes, exocytosis and/or erosion of dermal papilla).

Additionally, the biopsies were graded according to the inflammatory response in dermis as mild or absent (score 1), moderate (score 2), or (score 3) severe.

2.6. Fish

The biopsies were hybridized using oligonucleotide probes specific for Domain bacterium, Dichelobacter nodosus; Treponema phagedenis as well as Treponema spp.; (a Treponema group probe) as previously reported [8].

The hybridized sections were all read and scored by one of the senior authors. The total bacterial (probe for Domain bacterium) colonization of the epidermis was scored from 0 to 3 (0 = no invasive bacteria, 1 = low number of invasive bacteria, 2 = moderate number of bacteria, and 3 = high number of invasive bacteria). Similarly, the prevalence Treponema spp. and T. phagedenis-like organisms were scored from 0 to 3, whereas the prevalence of D. nodosus was scored 0 = no hybridization, or 1 = positive hybridization.

2.7. Collection of Fecal Samples and DNA Extraction

Forty eight samples of faeces were collected from the 8 heifers from herd 1 and 2 during 6 consecutive days (one sample per animal per day). The samples were frozen immediately and stored at –20˚C.

DNA was extracted from the samples using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany): 180 - 220 mg of frozen faeces was transferred to ASL lysis buffer and DNA was extracted following manufacturer’s protocol. The purified DNA was quantified (the yield per sample was approximately 6 µg with no variation between the samples) and diluted in nuclease-free water (Applied Biosystems, Foster City, CA, USA) to the concentration of 6.5 ng × µL^–1.

DNA for standard serial dilutions was obtained from a Treponema phagedenis isolate (isolate VI) provided by the Dept. of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden.

DNA was extracted using Easy-DNA Kit, protocol #3 (Invitrogen, Taastrup, Denmark).The quality of the purified DNA was verified both photometrically using a (NanoDrop 2000) spectrophotometer (Thermo Scientific, Wilmington. DE, USA) and by performing a PCR with general and Treponema spp. specific 16S RNA gene primers.

2.8. qPCR

The serial dilutions with well-defined number of DNA copies were created by cloning Treponema phagedenis 16S RNA gene fragment into pCR4Blunt-TOPO vector (Zero Blunt^® TOPO^® PCR Cloning Kit for Sequencing, Invitrogen)

PCR was performed in 4 × 50 µL reaction mixture containing: 0.5 µM of each primer, dNTP (each nucleotide 400 µM), MgSO₄ (2.0 mM), pfu DNA polymerase (0.05 U × µL^–1; Promega Corporation, Madison, WI, USA), template DNA from Treponema phagedenis isolate (40 ng per 50 µL of mix).

The cloning was performed according to the Invitrogen protocol for Zero Blunt^® TOPO^® PCR Cloning Kit. Five selected transformants were cultured overnight, harvested and the plasmids were purified using Plasmid Mini Kit (Qiagen, Cat. No.12123). The purified plasmids were quantified (Nanodrop) and digested with Pst I (Promega, cat. no. R4114) following Promega’s protocol. The plasmid digest was then purified with MinElute PCR Purification. The insert sequence was controlled by sequencing purified plasmid using the Treponema primers. Sequencing was performed on a 3130 Genetic Analyzer (Applied Biosystems, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, cat. no. 4337455) according to the manufacturer’s protocol. The samples were run in triplicates.

qPCR was performed using a Rotor-Gene 6000 (Corbett Research, Sydney, Australia).

For qPCR on DNA extracted from faeces qPCR was run as single reactions for standard dilution and as triplicates for the samples. The conditions of PCR were the same as described above.

2.9. Statistical Analyses

To determine whether clinical classification of lesion stage, changes in epidermis and, inflammatory response in dermis were influenced by the prevalence of Treponema spp., prevalence of T. phagedenis-like, and prevalence of D. nodosus a multinomial (ordered) regression model was employed. The model was fitted using generalized estimating equations (GEE) with a working independence covariance structure based on the specific lesion within cow in order to accommodate any effect of repeated observations on a single lesion. Misspecification of the correct correlation structure was not critical with respect to the fixed effect estimates and would result in a loss of efficiency. Clusters defined by leg within cow were considered to see if this influenced the standard error of the estimated parameters.

Due to the size of the dataset and the low number of animals with DD a forward selection scheme was used to identify the associated predictors and the biological effect of the associated predictors was subsequently quantified.

2.10. Generalized Estimation Equation

For the generalized estimating equation (GEE) procedure, a multinomial error distribution and a cumulative logistic link function was employed. Initially, a working independence covariance structure with clusters based on individual legs was used. However, clusters based on individual lesions or on the broader grouping defined by cows were also considered. Explanatory variables using a forward selection approach was included and subsequently the full model was reduced using model reduction.

3. Results

None of the 8 naïve heifers developed digital skin lesions during the 12 week observation period. The two heifers from tie stall (herd 1) developed hyperkeratosis of the digital skin above the heel bulbs. There were one case of lameness due to distortion and swelling of the fetlock joint on a foreleg. Otherwise the naïve heifers remained healthy. However, one biopsy was positive for Dichelobacter nodosus in the final biopsy sample collected from one of the healthy heifers at the end of the 12th week. The clinical evaluation of the skin showed hyperkeratosis and this was confirmed in the histology sample upon analysis.

Table 1 shows the status of DD lesions during the 12 weeks of study. The first lactation cow (herd 3) introduced after four weeks had DD on both hind legs. On left hind leg there was a lesion on an interdigital hyperplasia and a lesion between the heel bulbs. On the right hind leg there was a lesion between and above the heel bulbs. Both lesions between and above the heel bulbs healed completely, during the eight weeks in the experimental setting, where the legs were constantly covered in manure. However, the DD on the interdigital skin remained throughout the study. Three of the four heifers from herd 4, had lesions that healed completely during the study period.