Encephalitozoon cuniculi is a microsporidial parasite that primarily infects domestic rabbits (Oryctolagus cuniculus). It is the causative agent of encephalitozoonosis, a disease with an internationally recognized seroprevalence among rabbits. This study determines the presence, clinical manifestation, and serological status of encephalitozoonosis in pet rabbits in Slovenia using various diagnostic procedures. From 2017 to 2021, 224 pet rabbit sera were collected and tested for encephalitozoonosis with the indirect immunofluorescence assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against E. cuniculi were confirmed in 160 (65.6%) cases. Most seropositive rabbits suffered from neurological clinical signs or signs of gastrointestinal disorders such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia, and fewer showed clinical signs related to the urinary system or phacoclastic uveitis. A quarter of the positively tested rabbits presented without clinical signs. Hematological and biochemical blood analysis confirmed that seropositive animals had elevated globulin and deviated albumin levels in comparison to the normal reference values of non-infected animals. Furthermore, rabbits with neurological clinical signs showed statistically significant higher levels of globulins and total protein. Sixty-eight whole-body radiographs and thirty-two abdominal ultrasound reports were analyzed, looking for changes in the shape or size of the urinary bladder, presence of urinary sludge or uroliths, and any abnormalities related to the kidneys (shape, size, or nephrolites). The results suggest that neurological defects in the urinary bladder caused by E. cuniculi lead to a distended urinary bladder and consequently dysuria, incontinence, urine scalding, and sludgy urine.

Background: Encephalitozoon cuniculi is an obligate intracellular parasite belonging to the phylum Microsporidia, family Encephalitozoonidae. The parasite is distributed worldwide and infects a variety of mammals, with rabbits being the main host. Currently, the most reliable diagnostic method is serological testing, but various molecular techniques are also being used. Objective: This study assesses the presence, genotypes and clinical signs of E. cuniculi infection in pet rabbits in Slovenia, using molecular and serological methods. Methods: From January 2022 to 2024, urine samples from random pet rabbits ( n = 247) were collected for polymerase chain reaction (PCR) testing and transmission electron microscopy (TEM) to search for the presence of E. cuniculi genetic material or spores. Results: With the real‐time PCR, E. cuniculi genetic material was detected in 37 of 247 (14.98%) rabbit urine samples. Serological testing revealed specific immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies in 91 of 133 rabbits (68.42%). A quarter of the serologically positive rabbits (25.27%) and almost half of the molecularly positive rabbits (43.24%) were clinically healthy and showed no signs of encephalitozoonosis. E. cuniculi genetic material in urine was mostly discovered in rabbits with signs of urinary tract disorders (27.03%) or neurological clinical signs (21.62%). Genotype I of E. cuniculi was detected in all rabbits. Despite the detection of the pathogens’ DNA, no E. cuniculi spores were observed by TEM. Conclusion: E. cuniculi infection is common among pet rabbits in Slovenia, with a high seroprevalence and detectable urinary shedding of parasite DNA. The presence of clinically healthy but infected rabbits indicates that subclinical infections are frequent. Genotype I appears to be the predominant genotype in the studied population. Combined molecular and serological testing may improve the detection and epidemiological characterization of E. cuniculi infections in rabbits.