Equine Hoof Canker: Bovine Papillomavirus Infection Is Not Associated With Impaired Keratinocyte Differentiation.

Impaired keratinocyte differentiation has recently been suggested as a key event in equine hoof canker development. Koilocytotic appearance of keratinocytes, one of the most characteristic morphological alterations in hoof canker tissue, is also a common marker for papillomavirus (PV) infection, and bovine PV-1 and/or -2 (BPV-1/2) has previously been detected in equine canker patients. Therefore, the present study aimed to correlate the frequency and severity of koilocytotic keratinocytes with BPV detection in hoof canker samples. Hoof tissue of 5/18 canker-affected horses and 2/6 control horses tested positive for BPV-1/2 DNA using polymerase chain reaction. Thus, no association between the presence of BPV-1/2 papillomaviral DNA and koilocytotic appearance was found. Proteins associated with but not specific for PV infection were also investigated. Using immunohistochemistry, specific adhesion molecules (E-cadherin and ß-catenin) and intermediate filaments (keratins 6 and 14) important for intact epidermal barrier function and keratinocyte differentiation were documented in control samples (n = 6) and in hoof canker tissue samples (n = 19). Altered expression patterns of intermediate filaments and adhesion molecules were demonstrated in canker tissue, confirming the importance of incomplete keratinocyte differentiation, as well as the crucial role of keratinocyte differentiation in hoof canker.

Prevalence of bovine papillomavirus and Treponema DNA in bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is a common infectious foot disease whose aetiology is not fully understood. Its origin is thought to be multifactorial, with treponemes being involved. Using PCR-based techniques, BDD samples from 45 affected cows and intact skin from 8 BDD-affected and 33 healthy cows were assessed for the presence of bovine papillomavirus and Treponema DNA. BPV DNA (mainly BPV-1/2) was detected in 22% of lesions and one skin sample from affected animals, and in 15% (BPV-1/-2) and 23% (BPV-3/4/6/9/10) of skin samples from healthy cows. Using quantitative PCR, Treponema DNA was demonstrated in 38/45 BDD lesions, with bacterial DNA loads ranging between 2 × 10(3) and 2.78 × 10(5) copies/40 ng of total DNA. Qualitative PCR confirmed this result and revealed Treponema DNA in 4 additional BDD samples, thus leading to an overall infection rate of 93.3%. Sequence analysis of amplified Treponema DNA revealed T. pedis sp. nov. in 51%, T. medium ssp. bovis in 37.7%, and T. phagedenis ssp. vaccae in 4.4% of lesions. T. brennaborense was not detected in any of the samples. Six BDD samples contained type IV oral Treponema strains, 6 other harboured so far unpublished Treponema sequences. To our knowledge, this is the first report providing information on BPV infection in BDD-affected cattle, and the Treponema DNA load and occurrence of type IV treponemes in BDD samples. Our findings further support an etiologic association of treponemes, particularly T. pedis sp. nov., with BDD disease, yet indicate that BPVs do not directly contribute to BDD development.

Scanning electron microscopy and fungal culture of hoof horn from horses suffering from onychomycosis.

Horn samples were taken from the hooves of eight horses with clinical signs of equine onychomycosis in at least one hoof capsule. None of the horses had a documented mycological history. The predominant alterations of the horn capsules were sand cracks, white line disease, brittleness (especially around the nail holes), parakeratosis and bruising. The horn samples were stored in sterile tubes for transportation and transferred onto Sabouraud Dextrose Agar and dermatophyte test agar for mycological examination within 6 h. Fungal cultures were incubated for 30 days at room temperature. Fungal identification was based on colonial morphology and microscopic examination of conidia. Horn samples were also stored at -80°C until used for scanning electron microscopy (SEM). The fungal culture revealed that the hoof horn from all eight horses was infected with keratinophilic fungi. The keratinopathogenic fungi Trichophyton spp and Scopulariopsis brevicaulis were also detected in six horses. SEM revealed severe alterations of the horn structure in horn samples infected with keratinopathogenic fungi compared to horn samples from a sound hoof. The most evident changes were deterioration of the tubular structure of the horn wall, disruption of the horny layers, superficial lysis of cornified cells and the presence of fungal elements. Samples without dermatophyte or Scopulariopsis infection, in contrast, were similar to healthy hoof horn.

Is moisture really the most important influencing factor on the mechanical properties of claw horn? Modulus of elasticity and dry-matter content in flat and contracted claws.

The modulus of elasticity (E) and dry matter content (DMC) of horn samples from two types of claw deformity (contracted and flat claws) were determined according to EN-ISO 527 and ASTM D 638-03. The overall E values for all segments combined were 168 +/- 154 MPa (mean +/- SD, n = 79) and 248 +/- 196 (n = 90) for contracted and flat claws, respectively. These values were approximately 60% and 40% less than the E value of sound claw material. The high correlation between E and DMC, as previously found for sound claw horn material was not present in horn obtained from these types of pathologically altered claws.

Modulus of elasticity and dry-matter content of bovine claw horn affected by the changes of chronic laminitis.

The mechanical properties of horn samples from 22 hind claws with chronic laminitis were determined in adult Austrian Fleckvieh cows. The resistance to deformation was quantified as the modulus of elasticity (E). Tension tests revealed mean E values of 520MPa for the dorsal wall, 243MPa for the abaxial wall, 339MPa for the axial wall and 97MPa for the sole. E tended to be lower in laminitic horn than in sound horn in all segments tested, with the difference being largest in the abaxial wall. The mean dry-matter content (DMC) of the laminitic claws was 75.8% in the dorsal wall, 75.86% in the abaxial wall, 71.15% in the axial wall and 69.28% in the sole. These values are generally comparable to those for sound claws except in the axial wall. Further, E and DMC were only correlated in the axial wall. Chronic laminitis leads to a low resistance of claw horn to mechanical insults in the dorsal wall, abaxial wall and sole, and to the loss of a correlation between the E and DMC in these segments. The reason for these alterations is therefore not increased ingress of moisture, but must be due to changes in the microstructure, biochemical components and/or horn formation by the diseased dermis.

In vitro effects of fungi isolated from equine hooves on primary human keratinocytes.

The effects of two dermatophytes (Microsporum gypseum and Trichophyton mentagrophytes) and four moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living keratinocyte cultures were examined in vitro using primary human keratinocytes. Rates of apoptosis of infected cells were determined using a colorimetric TUNEL system which detects the characteristic nuclear DNA fragmentation of apoptotic cells. The cytotoxicity of the individual fungi was tested by quantitatively measuring cytosolic enzyme lactate dehydrogenase, released upon cell lysis, in culture supernatants. Additionally, the cell structures within the infected keratinocytes in cultures were examined by scanning electron microscopy. All of the fungi exhibited high cytotoxicity, whereas the development of only the two dermatophytes and the mould Scopulariopsis brevicaulis resulted in distinctly increased apoptosis. Electron microscopy showed that all fungi studied caused similar alterations in the cell structure, with Microsporum gypseum being the most harmful. Increasing loss of cell adhesion as a consequence of a decreasing number of reticulating cell appendices and a reduced cell plasticity were the most evident alterations.

In vitro degradation of equine keratin by dermatophytes and other keratinophilic fungi.

Keratinolytic properties of two dermatophytes (Microsporum gypseum, Trichophyton mentagrophytes) and three moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum) isolated from diseased equine hooves were examined to improve the understanding of pathogenic mechanisms leading to equine onychomycosis. Equine hoof horn material and skin, as well as hoof keratin and dermal keratin extracted from corresponding tissues, were used as sole carbon and nitrogen sources in five test tubes for each fungus. Within 18 days, supernatants of all tubes were repeatedly examined for keratinolytic activity by SDS-PAGE and Western blot analysis. In addition, fungal growth rates were determined to identify the preferred tissue of the individual fungi. Among the fungi examined, M. gypseum was the most keratinolytic species, followed by T. mentagrophytes and S. brevicaulis. In the concentration applied, the moulds A. alternata and G. candidum showed minimal keratinolytic activity. With respect to growth rates, M. gypseum favoured hoof horn material, S. brevicaulis and G. candidum preferred skin as a keratin source, whereas for the other two fungi no clear preference was detectable.