Encephalitozoon cuniculi spores in blood vessels in the brain of white New Zealand rabbits

Abstract

Encephalitozoon cuniculi is a zoonotic mycotic pathogen that infects rabbits. When the host becomes infected, spores in the brain parenchyma cause severe granulomatous meningoencephalitis. The mechanisms by which spores reach the CNS are poorly understood. We divided 27 white New Zealand rabbits into 2 groups: 15 infected orally with 10⁶ spores and 12 uninfected controls. Animals were euthanized at 30 d post-infection (dpi). Histologically, spores were present within brain blood vessels at 30 dpi. To our knowledge, the localization of free E. cuniculi spores has not been demonstrated previously within blood vessels in the brain of rabbits.

Microsporidia of the species Encephalitozoon cuniculi are zoonotic, opportunistic, single-celled, intracellular, and sporulated fungi that can infect vertebrates, including mammals, birds, and reptiles. ⁸ This pathogen causes severe granulomatous inflammation in the brain of the host. ¹⁰ After being ingested, E. cuniculi spores invade and colonize intestinal epithelia and macrophages. These infected inflammatory cells then can migrate to lymphatic or blood vessels, spreading the spores to various organs, including the kidneys and brain. ²

The clinical signs in infected animals are diarrhea, torticollis, ataxia, nystagmus, convulsions, circling, and death. ⁶ Typical microscopic lesions include severe interstitial nephritis and granulomatous meningoencephalitis. ⁷ Although the release of spores into the bloodstream seems to be widely accepted,^1-4 we did not retrieve any cases of E. cuniculi spores in the bloodstreams of rabbits in a search of PubMed, using the terms “Encephalitozoon”, “spore”, “brain”, “rabbit”, “blood vessel”, and/or “microsporidia”, suggesting that this condition has not been reported previously.

We obtained twenty-seven 2-mo-old white New Zealand rabbits, both male and female, from the Small Species Center (Tlaxcala, Mexico). Animals were clinically healthy and seronegative for E. cuniculi. Fifteen rabbits were infected PO with 10⁶ viable E. cuniculi spores (ATCC 50789) in 1 mL of saline solution. Twelve uninfected animals were used as negative controls and received only 1 mL of saline solution PO. All animals were housed at the Biological Models Unit of the Center for Research and Development in Health Sciences at the Universidad Autonoma de Nuevo Leon (UANL; Escobedo, México). Confinement conditions, food, and water adhered to the Official Mexican Standard (NOM-062-ZOO-1999) for the use and care of laboratory animals, and all animal protocols were approved by the Facultad de Medicina Veterinaria y Zootecnia-UANL (FMVZ-UANL) Bioethics and Animal Welfare Committee (09/2019).

Thirty days after infection, all animals were sedated with 15 mg/kg/IM tiletamine/zolazepam (Zoletil 100; Virbac) and euthanized by intravenous overdose with pentobarbital sodium (180 mg/kg/IV; Laboratories Aranda). Samples from the brain were collected, fixed in 4% paraformaldehyde, and prepared for routine histologic analysis (H&E and Nissl staining; Sigma) and immunohistochemistry (IHC; Abcam). For the IHC assay, serial 4-µm brain sections were obtained, deparaffinized, and rehydrated in graded ethanol, dH₂O, and PBS. Heat-induced antigen retrieval was performed with citrate buffer (pH 6.0) at 80°C for 80 min. Nonspecific binding sites were blocked with 10% normal goat serum and endogenous peroxidase was quenched with 3% hydrogen peroxide. A mouse anti–E. cuniculi monoclonal antibody (mAb; 1:100 dilution; Bordier Affinity Products) then was incubated overnight at 4°C. Primary antibody was then detected with a goat anti-mouse IgG antibody, incubated at room temperature for 30 min. Positive immunoreactions were visualized (HRP/DAB detection system ab64264; Abcam). Slides were counterstained with hematoxylin and mounted with resin. Positive and negative control sections were processed simultaneously.

Histologically, we observed prominent granulomas both in the white and gray matter of all infected animals, specifically in the telencephalon and rhombencephalon ( Fig. 1A ). We also observed E. cuniculi spores within blood vessel lumens in the brain (Fig. 1B, 1C). In these same anatomic regions, intravascular spores were observed as oval-or-pyriform, 1.0–2.0-μm microorganisms, alone or in clusters ( Fig. 1D ). Microsporidian cysts in the thalamus were only observed in 3 of the 15 infected rabbits. Interestingly, phagocytic cells were not observed in the intravascular space nor in cysts. The brain parenchyma was marked by gliosis, edema, necrosis around granulomas, and cysts. None of the uninfected controls developed brain lesions. We did not evaluate other tissues, including the kidney, liver, and lungs, microscopically.

Figure 1.

Encephalitozoon cuniculi in white New Zealand rabbits. A. Microsporidian cerebral granuloma. H&E. Bar = 50 μm. B. Microsporidian spores (arrow) within a blood vessel and free in the brain parenchyma. Nissl stain. Bar = 10 μm. C. E. cuniculi–positive spores (arrow) within a brain blood vessel. Lumen (L). Nuclei of endothelial cells (dashed arrows). Bar = 10 μm. D. Brain cyst (arrow) containing immunopositive E. cuniculi spores, some of which were free in brain parenchyma (arrowhead). Bar = 10 μm.

The morphologic details of the spores and the positive immunolabeling of the microsporidian allowed us to identify the parasite, which we assumed was traveling freely within the blood vessels. We report the early detection of spores within brain blood vessels of rabbits at 30 dpi. This suggests that, following ingestion, E. cuniculi intact spores enter the bloodstream by an undetermined mechanism, ⁴ allowing them to spread to other organs, such as the brain, establishing in parasitic cysts and forming granulomas. ⁹ The mechanism by which E. cuniculi spores cross the blood-brain barrier (BBB) remains unknown. However, the parasites could use derived molecules, such as adhesins and proteases, to promote BBB penetration, ³ a mechanism that has been reported to occur in Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium falciparum infections. ⁵ These microorganisms share similarities with microsporidians, because they are intracellular, eukaryotic, single-celled parasites, travel via the bloodstream, and infect the brain as part of their life cycle. Although literature regarding E. cuniculi implies host dissemination via leukocytes ⁸ ; to date, microscopic evidence is lacking.

Our study clearly identified free spores within the bloodstream of rabbits. From our experimental animal model, we conclude that host dissemination not only occurs with the help of “cell transporters,” but also via spores traveling freely within blood-lymph routes—an anatomic environment that appears to favor spore survival. Future studies focusing on the bloodstream at different times post-infection may elucidate the exact mechanism of entrance into the brain via the BBB.