In December 2015 two Pedigree Limousin calves (1 male and 1 female) were presented to the University Veterinary Hospital (UVH) at University College Dublin (UCD). Both were full siblings born to recipient heifers from implantation via embryo transfer. The two calves presented two days apart. The first (Calf A) was a 12-h-old bull calf born unassisted but from birth remained in lateral recumbency and was unable to maintain a normal posture even when assisted. The calf was non-responsive and appeared grossly abnormal, as the skin over the thorax and abdomen was moist (Fig. 1a). The orbital fissures were almond shaped with bilateral clouding of the cornea (Fig. 1b). The calf had thick folds of cervical skin and, on closer inspection, had multiple small fissures in the skin over large parts of the body, particularly over joints and the medial aspect of the hindlimbs (Fig. 1c). Whilst examining the skin more cracks appeared even following gentle manipulation. Additionally this calf displayed involuntary muscle tremors that had been occurring since birth. The calf was euthanised and submitted for post mortem examination. Two days later a second Pedigree Limousin calf (Calf B) presented to the UVH, a female with similar clinical features as Calf A. In addition, Calf B had a ventromedial strabismus bilaterally and pale mucous membranes. This animal was also euthanised and submitted for post mortem examination. Both calves were euthanised using intravenous pentobarbital sodium (Release, 5 ml/10KG BW).
Post-mortem examination revealed the skin of Calf A to be diffusely moderately thickened, with linear fissures noted over the lateral neck, dorsum, and on the flexor surface of the limb joints, axillae, and groin. Occasionally the underlying dermis was exposed. The skin was diffusely damp; the oral mucous membranes were red. Bilaterally, the second incisors were small and subgingival. Calf B had similarly thickened, damp skin, with loose fragments of crusty skin in the regions where the fissures were seen in Calf A. Internal organs of both calves appeared normal; however, there were focally-extensive haemorrhages in the abomasal mucosa of Calf A, with similar, smaller, lesions in Calf B. In both animals the abomasum contained large amounts of clotted milk, but the remainder of the gastrointestinal tract were empty. In addition, there was a small focus of tortuous, dilated blood vessels on the diaphragmatic surface of the spleen of Calf A. All limb joints of Calf B contained a small amount of serosanguinous fluid.
Multiple tissues were sampled from the carcasses for histopathologic processing. Briefly, tissues were fixed in 10 % formalin, dehydrated in alcohol, embedded with wax, cut into 5 μm sections, and mounted on glass slides. All tissues were stained with haematoxylin and eosin (H&E). A selection of skin samples were also stained with periodic acid schiff (PAS), Masson’s Trichrome, and Alcian blue using routine protocols. The Alcian blue stain was employed to look for acid polysaccharides which are found in the ground substance of skin, there was no increase in ground substance in these cases. Tissues were examined using light microscopy by pathologists at UVH UCD and the Joint Pathology Centre, Maryland, U.S.A.
Histologically, the skin of Calves A and B exhibited a marked diffuse reduction in the density and number of mature collagen fibres, preferentially affecting the superficial dermis. The remaining fibres were short, fine, pale, fibrillated and fragmented. Trichrome staining by the Masson’s method further highlighted these abnormalities, confirming the dysplastic nature of the collagen. Collagen fibres of the deep dermis appeared relatively spared. Calf B exhibited similar, but less severe, changes (Figs. 2d; e). Ultrastructurally, abnormal dermal collagen fibres of the haired skin of the back consisted of irregular fibrils arranged in loosely woven, twisted flat, and/or helical ribbons with irregularly spaced or lost transverse striations (Fig. 3).
Based on the clinical presentation and the characteristic histological and transmission electron microscopy findings, a diagnosis of dermatosparaxis was made. Given the previously characterised gene mutation reported in Belgian Blue cattle [6], skin samples were examined to discern if the same mutation was responsible in the current case. 5 animals were tested, Calf A, Calf B and three healthy controls (Holstein-Friesian calves). DNA was extracted from fresh frozen skin tissue using the Qiagen DNeasy Blood & Tissue Kit as per the manufacturer’s instructions; DNA quantity was assessed using the NanodropTM spectrophotometer. A primer pair as previously described [17] was used to detect a 17 bp deletion within the coding region of the ADAMTS2 gene;
Ten nanogram of DNA was used for each PCR, reactions were performed in 25 μl and contained 17.5 μl water, 1 AmpliTaq Gold buffer, 2 μl primer mix, 1.5 mM MgCl2, 0.5 μl dNTP mix and 1U of AmpliTaq Gold. The thermal cycling conditions consisted of an initial activation step of 95 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 56 °C for 45 s and 72 °C for 1 min with a final elongation step of 72 °C for 5 min. PCR products were analysed using the Agilent DNA 1000 chip. The PCR product from all DNA samples, both cases and controls, yielded a single sharp band at the expected size of 101 bp, and no deletions, indicating that the genetic defect in these animals is not the same as described previously [6].
The clinical and pathological features described in these two calves are consistent with a diagnosis of dermatosparaxis [1, 2, 16, 17] and is the first reported case of this disease in this breed. The skin fragility and hyperhidrosis, along with the marked fragmentation and depletion of superficial dermal collagen, are consistent with previous bovine cases [1]. Interestingly, the PCR testing carried out in this case did not find a mutation in the ADAMTS gene described previously in cattle. [6, 15] and was similar to the absence of this mutation in Drackensberger cattle [17] highlighting the fact that a range of mutations can result in a broadly similar disease phenotype [5]. Inbreeding has previously been incriminated as the underlying mode of inheritance in Belgian Blue cattle [2] and in the Charolais breed [18]. In the current case, there is common parentage in the ancestry; the dam’s great grandsire and the sire’s grandsire were the same bull.
