Morsy et al. Acta Vet Scand (2020) 62:11 https://doi.org/10.1186/s13028-020-0509-6 Acta Veterinaria Scandinavica RESEARCH Open Access Encephalitozoon cuniculi infection in farmed rabbits in Egypt Eman Anter Morsy1, Heba Mohammed Salem1, Marwa Salah Khattab2, Dalia Anwar Hamza3 and Mai Mohammed Abuowarda4* Abstract Background: Encephalitozoon cuniculi is an important microsporidian parasite with zoonotic potential. The pre- sent study highlights the impact of encephalitozoonosis on rabbit health in Egypt. Three rabbit farms in Giza, with a total of 16,400 rabbits were investigated due to occurrence of rabbits displaying clinical signs consistent with encephalitozoonosis. Results: Clinical signs observed during a 4 months observation period in 2018 included vestibular disease, pare- sis, limb paralysis, cataracts, phacoclastic uveitis, frequent urination, marked decrease in body weight and in some pregnant females, also repeated abortions. The total morbidity rates in adult and young rabbits were 76.7% and 81.5%, respectively. The highest mortality rate was recorded in ofspring (12.3%), followed by dams (5.6%), and the lowest recorded mortality rate was in males (0.04%). Post-mortem fndings included enteritis, pale enlarged kidneys, congested leptomeninges, focal brain necrosis, and endometrial congestion. Histopathological examination revealed nonsuppurative meningoencephalitis and glial nodules with central necrosis in the brain, vacuolation and necrosis of renal tubular epithelium, and corneal ulceration and ruptured lens capsule with fragmentation of lenticular fbres. E. cuniculi were observed in the brain, retinal ganglion cells, kidneys, and liver. Transmission electron microscopy exami- nation revealed the presence of diferent developmental stages of E. cuniculi in the brain and kidney. Presence of E. cuniculi was confrmed by conventional polymerase chain reaction using a universal 16S gene for Encephalitozoon spp. followed by sequencing and sequence analysis. Conclusions: The presence of E. cuniculi in rabbits was confrmed at three farms in Egypt. Nervous signs and ocular lesions were the most predominant fndings in these farms. Keywords: Encephalitozoon cuniculi, Histopathology, Incidence, Polymerase chain reaction, Rabbit, Transmission electron microscopy Background as some protists [1]. E. cuniculi, which was frst identi- Encephalitozoon cuniculi is an obligate intracellular fed in 1922 in a colony of research rabbits in the USA, spore-forming protozoan parasite belonging to the phy- is now recognized as a common pathogen and a signif- lum Microsporidia, which contains approximately 1400 cant cause of disease among pet rabbits [2–4]. E. cuniculi species distributed into about 200 genera. E. cuniculi can has a zoonotic potential, especially for immunocompro- infect almost all invertebrates and vertebrates, as well mised adults and for children [5]. Terefore, E. cuniculi has received increased attention [6], and hygienic precau- tions should be undertaken when humans are in contact *Correspondence: [email protected] 4 Department of Parasitology, Faculty of Veterinary Medicine, Cairo with rabbits or rabbit products [7]. University, PO Box 12211, Giza, Egypt Te life cycle of E. cuniculi takes 3 to 5 weeks to com- Full list of author information is available at the end of the article plete [8]. Hosts are infected by ingestion or inhalation of © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Morsy et al. Acta Vet Scand (2020) 62:11 Page 2 of 11 spores or by transplacental transmission [9]. After inges- importance [25]. Parasites are responsible for direct and tion, the spores invade enterocytes and then enter the indirect losses of rabbits that are attributed to acute ill- bloodstream or the lymphatic system, possibly through ness and death, premature slaughter, decreased growth Peyer’s patches or interepithelial lymphocytes, and there- rate, weight loss and late maturity of slaughter stock [26]. after reach organs such as the brain, kidney and liver. In Egypt, E. cuniculi was not reported until 2011, when Spores can also transmigrate after having been ingested its presence was indicated by serological screening of the by phagocytes present in the intestinal mucosa [10]. domestic rabbit population in northern Egypt, in Behera, Infected rabbits develop serum antibodies generally Alexandria and Khafr El-Sheikh for E. cuniculi and Toxo- within 3 weeks post-exposure and excrete E. cuniculi by plasma gondii [7]. However morphometric or pathologi- 6 weeks [11]. Te spores are excreted through urine and cal investigations have not yet been carried out. faeces; hence, transmission generally takes place after the Te purposes of this study were (1) to investigate ingestion of water or food contaminated with infective encephalitozoonosis in rabbit farms in middle Egypt; spores [5]. (2) to confrm the presence of E. cuniculi using pathol- Encephalitozoon cuniculi infection of the central nerv- ogy and molecular techniques and (3) to describe clini- ous system is associated with development of vestibular cal signs, histopathological changes, morphology features disease, which is characterized by torticollis, ataxia, pare- and genetic characteristics of the parasite. sis, nystagmus, seizures, and longitudinal rolling and the animal often becomes recumbent [12, 13]. In addition to Methods nervous signs, infected rabbits develop eye lesions and Animals chronic renal failure [14]. In the eye, infammation of the Tree commercial rabbit farms (A–C) in the Giza gov- anterior lens capsule can lead to its spontaneous rupture. ernorate in middle Egypt, with a total of 16,400 rabbits Moreover, phacoclastic uveitis develops because of peri- (4121, 5583 and 6696 rabbits for each of the three farms, lenticular fbroplasia together with lens capsule rupture respectively) founded the study population. Rabbits on [15, 16]. Kidney lesions can be observed within 4 weeks, these farms were investigated during a 4 months period and brain lesions within 8 weeks after infection [17]. in 2018. Microscopic lesions in the brain and kidney, consist of Rabbits that exhibited clinical signs suggestive of E. focal non-suppurative granulomatous meningoencepha- cuniculi infection (n = 13,242) underwent a clinical litis and focal to segmental interstitial nephritis with var- examination. Of these 13,242 rabbits, 2680, 4658 and ying degrees of fbrosis [18]. 5904 rabbits came from farms A, B and C, respectively. Transmission electron microscopy (TEM) can be Rabbits of diferent breeds, including New Zealand, used to confrm encephalitozoonosis by detecting the Chinchilla, and Californian, were reared in a wire net polar flament within spores and demonstrating ultra- cage system and fed commercial pellets. Rabbit handling structural features. TEM, along with newly applied procedures were performed in accordance with the appli- molecular approaches, can contribute to the taxonomic cable legislation of the Institutional Animal Care and Use organization of the microsporidia [19, 20]. Polymerase Committee of the Faculty of Veterinary Medicine, Cairo chain reaction (PCR)-based methods that typically utilize University, Egypt (VetCU11112018015). primers for amplifcation of microsporidial rDNA have Te examined animals were classifed into two age been commonly utilized to enhance diagnostic sensitivity groups, i.e. young and adults. Young rabbits (n = 11,170) and specifcity; however, they are still not routinely used were from 0 to 4 months old, whereas adults (n = 2072) in diagnostic laboratories [21]. Te E. cuniculi genome were rabbits older than 4 months and up to 1 year old. Of is very small being only 2.9 Mb large and compressed. It the adult rabbits, 332 were males and 1740 females. harbours 2000 genes that are located within minute inter- genic regions (the mean intergenic region is 80 bp) [22]. Clinical examination Analyses of genome diversity have revealed the existence Te rabbits were visually assessed from a distance and of four genotypes of E. cuniculi (EcI, II, III and IV) [23]. any abnormal behaviour was recorded. Each visual Rabbit production recently has become well estab- assessment took about 5 min/cage. If abnormal behav- lished in Egypt, but little is known about the exact rabbit iour was observed, the rabbit was taken out of the cage numbers. Most of the rabbit population in Egypt is in the and examined according to [27]. Tis consisted of inspec- hands of smallholders, while the rest belongs to the com- tion of the head, including ears, eyes,