**Background:** Besnoitiosis is a parasitic disease caused by *Besnoitia* spp., cyst-forming coccidia closely related to *Toxoplasma gondii* and *Neospora* spp. *B. bennetti* is specific to equids and forms chronic intracytoplasmic cysts in mesenchymal cells, mainly fibroblasts, in the skin, sclera, and mucosa. Clinical signs range from mild to severe debilitating disease. The life cycle and transmission mode remain unknown, with no definitive host identified. In Europe, sporadic cases have been reported since 2013, with *B. bennetti* confirmed molecularly in only one British and one Belgian donkey prior to this study. This report describes the first cases of *B. bennetti* in Irish donkeys, comparing findings to recent European cases.
**Methods:** Two donkeys from the same farm in southwest Ireland were investigated. Case 1 was a 2.5-year-old jenny with a suspect sarcoid on the muzzle; the mass was surgically removed and submitted for histopathology. Case 2 was a 2-year-old gelding with nodular dermatitis of the muzzle; six skin biopsies were taken. Both animals underwent hematology, biochemistry, and serological testing (ELISA, IFAT, Western blot) using *B. besnoiti* antigens due to cross-reactivity. Molecular analysis included ITS-1 PCR and sequencing, and microsatellite typing of six loci (Bt-5, Bt-6, Bt-7, Bt-9, Bt-20, Bt-21) from formalin-fixed paraffin-embedded skin tissue. Treatment with trimethoprim sulfadiazine (30 mg/kg PO BID for 30 days) was initiated for both.
**Key Results:** Histopathology revealed protozoal tissue cysts in the dermis: in Case 1, 31 cysts per cm² (mean length 315 µm, range 172–455 µm; mean width 247 µm, range 140–344 µm); in Case 2, 3–4 cysts per biopsy (mean length 410 µm, range 274–562 µm; mean width 314 µm, range 155–449 µm). Cysts had a characteristic three-layered wall (hyaline outer layer, host cell cytoplasm with multiple nuclei, inner membrane enclosing bradyzoites). Pericystic inflammation consisted of lymphocytes, macrophages, and few eosinophils. Case 2 also showed dystrophic calcification (calcinosis circumscripta). Both donkeys had scleral and conjunctival cysts (0.5–2.5 mm) and dermal cysts on the muzzle. Serology: ELISA S/P% values were 72.1 (Case 1) and 128.9 (Case 2); IFAT titers were 1:400 and 1:1600, respectively; Western blot confirmed *Besnoitia* spp. antibodies. Molecular analysis: ITS-1 PCR positive (mean Cq 25.9 and 31.2; Tm 81.0 and 81.4); sequencing showed 100% identity with *B. bennetti* and a T insertion at position 148. Microsatellite typing revealed identical patterns for both cases (Bt-5:12, Bt-6:13, Bt-7:9, Bt-9:8, Bt-20:8, Bt-21:6), matching UK *B. bennetti* isolates but differing from North American isolates at Bt-7 (9 vs. 8 repeats). Treatment with trimethoprim sulfadiazine for 30 days did not result in clinical improvement of cysts.
**Clinical Implications:** This is the first report of *B. bennetti* in Ireland, confirming the spread of equine besnoitiosis as an emerging disease in Europe. The infection was identified in young, systemically healthy donkeys, with mild dermal signs and normal bloodwork, contrasting with severe cases reported elsewhere that often had comorbidities. The presence of cysts adjacent to a sarcoid in Case 1 and the association with old scars suggests possible transmission through skin trauma or insect vectors. The lack of response to trimethoprim sulfadiazine highlights the need for effective treatments. Besnoitiosis should be included as a differential diagnosis for chronic skin disease in donkeys, particularly for cutaneous masses, non-pruritic dermatitis, and treatment-resistant dermatitis. Histopathology is key for initial diagnosis, but molecular methods (PCR, sequencing, microsatellite typing) are essential for species confirmation. Future studies should investigate prevalence, transmission, and clinical impact in Irish donkeys and across Europe.