Metrics details Besnoitiosis is caused by different species of intracellular protozoan parasites belonging to the family Sarcocystidae and affecting multiple host species worldwide ten species are described infecting animals donkeys and zebras and was described in Africa and in the USA where donkey besnoitiosis is considered as an emerging disease A two-year-old male donkey was purchased in May 2016 in poor body condition (cachexia alopetic areas and pruritus mainly on neck and head) by the present owner in Le Roeulx (Belgium) from a milk producing donkey farm in Frasnes-lez-Buissenal (Belgium) hyperkeratosis (both flanks and neck) anorexia and cachexia A treatment with phoxim was given with no improvement A cutaneous biopsy of hyperkeratotic skin was performed in July It showed a perivascular eosinophilic infiltrate with a large thick walled cyst located in the dermis containing numerous bradyzoites This was highly suggestive of besnoitiosis Several skin biopsy samples were obtained for qPCR analysis and confirmed the presence of Besnoitia spp Further laboratory diagnosis tests were performed (western blot and rDNA sequencing) confirming Besnoitia bennetti aetiology for the male haematology and qPCR were negatives but the western blot showed the presence of antibodies directed to Besnoitia spp Further clinical examination performed in August highlighted scleral pinhead sized cysts (pearl) in the right eye and between nares Another ten-year-old female donkey purchased in France and sharing the same accommodation showed a good clinical condition but a thorough clinical examination showed the presence of numerous cysts on the inner face of upper labial mucosa A daily treatment based on sulfamethaxzole and trimethoprim (Emdotrim 60% Mix® 30 mg/kg) was given orally and some improvement was noticed This is the first evidence of Besnoitia bennetti infection (Protozoa: Apicomplexa) in donkeys (Equus asinus) in Belgium we describe two cases of besnoitiosis in donkeys a private veterinarian referred a donkey characterised by poor body condition and chronic skin lesions to the Faculty of Veterinary Medicine of Liège (Belgium) Patient’s skin lesions: a alopetic and crusty area on the right shoulder Shortly after its purchase, the animal was shorn revealing crusts and hyperkeratosis (on both flanks and the neck). The animal was anorexic and in poor body condition. A closer clinical examination in August highlighted scleral pinhead sized cysts (pearl) in the right eye and between nares (Fig. 2). The rest of the examination was unremarkable. Scleral pin-head sized cysts (pearl; red arrow) between nares Another ten year old female donkey (Grand noir du Berry breed) purchased several years ago in France (Loire region) by the same owner and sharing the same accommodation further clinical examination showed the presence of numerous cysts on the inner face of upper labial mucosa The two donkeys were kept in a fenced area (below 1 ha) The animals were fed a standard donkey food regimen composed of hay supplemented with protein-containing grain (oats) and occasional fruits and vegetables Previous medical treatments included routine vaccinations and prophylactic deworming (based on ivermectin) chickens and rodents) had free access to the farm and the paddock buildings Numerous flies were observed in the paddocks (Stomoxys calcitrans and Musca spp.) Skin scrapings were performed in different places (mainly flanks and neck where the lesions were the most obvious) on both animals A culture on Sabouraud dextrose agar-chloramphenicol (0.5 per 1000 m/w for three weeks at 37°C) yielded a negative result for ringworm Microscopic examination of the hair revealed neither fungal spores nor ectoparasites A Giemsa staining of an impression smear also gave negative results Several skin biopsies were taken using an 8 mm biopsy punch Histopathology and DNA extraction for qPCR and PCR were performed on those skin samples the material was stored in 10% phosphate-buffered formalin for histological examination and in 70% ethanol for molecular analyses The formalin-fixed sample was bisected and embedded in paraffin wax at 56 °C cut and stained with haematoxylin and eosin for routine evaluation This technique was performed by a veterinary diagnostic laboratory (Medvet Histopathological preparation from the male donkey showing a cyst full of bradyzoites (haematoxylin eosin; 10×) Several blood samples were taken for haematology and biochemistry analysis from both patients A blood sample from the male gave unremarkable results except a light anaemia [haemoglobin 10.1 d/l (normal range 11–19 d/l) red blood cells 5.22 106/mm3 (normal range 6.5–12.5 106/mm3) an eosinophilia [15.5%; 1631/mm3 (normal range < 5% and < 500/mm3 respectively)] and an increased gamma globulin fraction [32.8%; 23.9 g/l (normal range 13–21% and 5.5–19 g/l A blood sample from the female revealed only an eosinophilia (12.7% Lane -: sera from uninfected cattle; Lane +: sera from infected cattle; Lane 1: sera from uninfected donkey; Lane 2: male serum (4/8/2016); Lane 3: female serum (4/8/2016); Lane 4: male serum (9/9/2016); Lane 5: female serum (9/9/2016) The skin sample from the male was highly positive with a Ct of 25 but no Besnoitia DNA was detected in skin sample from the female Phylogenetic tree based on 938 bp rDNA sequence (containing complete sequences of 5.8S gene and ITS1 and ITS2 fragments and partial sequences of 18S and 28S genes) reconstructed using NJ/MP/ML methods showing the position of the isolated Belgian strain (GenBank: MG652473) and other Besnoitia from cattle and goat deposited in the GenBank database Bootstrap support (1000 replicates) is shown at each node as NJ/MP/ML; bootstrap values below 60% are not shown The scale-bar indicates the number of substitutions per site bennetti isolated from the equid host (donkey) was a distinct species to Besnoitia species (B Taking all these results a diagnosis of besnoitiosis was established in both animals A daily treatment based on sulfamethoxazole and trimethoprim (Emdotrim 60% Mix® 30 mg/kg) was given orally to the affected animal and some improvement was noticed the affected animal was treated for four months with sulfamethoxazole and trimethoprim alongside a discontinued hepatoprotective treatment (Sédochol®) The animal gained weight and no more skin conditions were visible besnoiti will perhaps provide new molecular targets useful for species phylogeny and genetic population structure very little data exists in the literature and these parameters do not seem to be of valuable assistance for diagnosis it could be useful for a correct evaluation of renal function before sulfamethoxazole-trimethoprim treatment neither isolation of parasites nor further molecular genotyping were achieved to determine the accurate Besnoitia species involved in these three studies This first report of besnoitiosis in donkeys in Belgium suggests an urgent need for an extensive serological survey to assess prevalence in not only Belgian donkey populations but also in France due to the geographical origin of the mare Difficulties in confirming diagnoses and the absence of efficient treatment options are probably responsible for the underreporting of besnoitiosis in European donkeys This report aimed to highlight to clinicians the necessity to include besnoitiosis in the differential diagnosis of chronic dermatitis unresponsive to routine topical and systemic treatments Whether this finding represents an unusual cluster of infections or reflects a wider distribution of subclinical infections which have largely gone undetected to date requires further study Donkeys are increasing in number and particularly in developing countries [36] Widespread distribution of this infection would be of international concern and veterinary medical importance This case report is the first unequivocal report of B bennetti infection of donkey in Europe based on clinical Further investigations are necessary to unravel the phylogeny of B bennetti and its epidemiology in Europe and elsewhere Bovine besnoitiosis: an emerging disease in Europe taxonomy and epidemiology of Besnoitia species (Protozoa: Sarcocystidae) A century of bovine besnoitiosis: an unknown disease re-emerging in Europe The role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle Comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against Besnoitia besnoiti tachyzoites and bradyzoites in bovine sera A 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kindness and help in handling the patients and Amelia Coggon for review and improvement of the English language The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request Laboratoire de Parasitologie et Maladies Parasitaires Caroline Vanvinckenroye & Yannick Caron FP and SS performed serology and molecular biology IT and CV performed the clinical part of the study SV performed the histopathological part of the study BL and MF read and improved the manuscript All authors read and approved the final manuscript The authors declare that they have no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1186/s13071-018-2993-3 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article