A male foal coming from a horse farm with 25 animals was born at term with eutocic delivery. The foal, at birth, showed the presence of lesions affecting the distal extremities of all four legs. From the carpus of the left foreleg and from the coronet of the other three legs, the skin was missing and the denuded dermis was covered by debris (Figure 1). No lesions were observed at the mucocutaneous junctions or in oral mucosa. The foal was treated with IV antibiotics (cefquinome 1 mg/kg twice a day and amikacin 15 mg/kg once a day) and a hoof boot was applied to the foot that had lost the hoof. Two days later, four skin biopsies were taken with the owner’s consent from the dorsal and palmar surface of the carpus, from the coronary band and from the coronet at the transition between affected and unaffected areas. Despite treatment, the foal died after 6 days and the owner declined the necropsy. Histologic examination of skin biopsies revealed the presence of vesiculo-bullous lesions characterized by a complete separation of the epidermis from the dermis at the level of the dermoepidermal junction. The blister formation also involved the infundibular portion of hair follicles and ulcerated areas were covered by thick serocellular crusts. Periodic acid-Schiff (PAS) staining revealed a PAS positive lamina densa at the pavement of the blister, attached to the underlying dermis. The macroscopic and histologic lesions were compatible with a hereditary epidermolysis bullosa. According to the PAS staining results, our case was compatible with a junctional epidermolysis bullosa (Figure 2). Transmission electron microscopy from formalin fixed skin biopsies confirmed the presence of a splitting between the epidermis and dermis. Basal keratinocytes were intact and demonstrated normal desmosomes but no hemidesmosomes were identifiable. The lamina densa was present on the pavement, at the dermal side of the blister, consistent with a splitting at the level of the lamina lucida (Figure 3).
Based on the macroscopic and histologic findings as well as the ultrastructural features, a diagnosis of hereditary junctional epidermolysis bullosa was made. Molecular tests aimed at the detection of known mutations associated with the disease, involving the Laminin 5 protein complex, were performed in order to confirm the diagnosis. Nucleic acids were extracted from 200 μl of total blood using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s instructions. Since the disease has a Mendelian autosomal recessive inheritance, foal’s DNA together with the DNA of the mother (admixed horse), of the father (heavy horse who was also the grandfather) and of the maternal grandmother (light horse) was tested at the cited loci. PCR was performed as previously described [4,8] using 30 ng of DNA as template for the amplification of LAMC2 and LAMA3 regions where the known mutations rely, and amplicons directly sequenced. PCR results were negative for LAMA3 deletion in all samples. The affected foal was homozygous for 1368insC in LAMC2 whereas the sire and the dam were heterozygous for the insertion (Figure 4).
These results confirmed that the mutation causing junctional epidermolysis bullosa in the foal was localized in the LAMC2, as already described in northern Europe’s coldblood breeds (Belgian Horse, Trait Breton and Trait Comtois) [4,10], which participated, with some lines, to the creation of Italian draft horses [11]; since the disease has a classical autosomal recessive Mendelian inheritance, both parents must be heterozygous (carriers).
Inbreeding, enhanced by erroneous breeding practices, should always be avoided as it can increase the frequency of potentially deleterious recessive alleles in the population and their phenotypic manifestation at individual level [12].