Encephalitozoon cuniculi–Associated Equine Encephalitis: A Case Report

Abstract

A case of encephalitis of unknown origin in the horse was investigated. Postmortem examination findings revealed a nonsuppurative granulomatous meningoencephalitis in the right hemisphere of the cerebral cortex. Testing for West Nile virus, equine herpes virus, equine infectious anemia, Toxoplasma gondii, Neospora caninum, and Sarcocystis neurona were negative. The horse had a titer for Encephalitozoon cuniculi, and sections from the affected area of the brain tested positive for the organism using both polymerase chain reaction (PCR) and immunohistochemistry. Amplicons generated using PCR were sequenced, and E. cuniculi genotype II was identified. This is the first case of E. cuniculi genotype II associated with encephalitis in the horse.

1. Introduction

Encephalitozoon cuniculi, a member of the phylum microsporidia, is a unicellular, obligate intracellular, spore-forming parasite [1–3]. Originally, microsporidia were classified with the protozoa; however, they were recently reclassified with the fungi [1,2,4]. Encephalitozoon cuniculi has a wide host range including rodents, domestic and wild canidae, cats, goats, cattle, horses, fish, insects, ticks, and nonhuman primates and is best described in rabbits [1–3,5,6]. Encephalitozoon cuniculi is emerging as a significant opportunistic infection of immune-compromised humans; it is also recognized as an emerging zoonotic risk [1,2,6–8].

Encephalitozoonosis is often not clinically apparent in the rabbit host. When it occurs, clinical disease usually manifests as encephalitis, uveitis, or nephritis [1–3,6]. In other species, E. cuniculi has been associated with encephalitis, abortion and/or placentitis, colic, and pyrexia [3,6]. Lesions are normally focal nonsuppurative granulomatous responses with perivascular cuffing [2,6,9]. The organisms can be demonstrated both intracellularly and extracellularly using Ziehl Neilson (ZN), Gram, and modified trichrome stains as well as immunohistochemistry [1,2].

Encephalitozoon cuniculi has been reported as a cause of placentitis and abortion in the horse, and serologic evidence of infection has been documented in both Israel and Brazil [3,5,6]. This article describes a case of equine encephalitis associated with E. cuniculi infection.

2. Case History

A 5-year-old National Hunt Thoroughbred gelding in training initially presented with weight loss and lethargy. Over the course of 9 days, clinical signs progressed from initial reluctance to move to abnormal placement of feet when walking, stumbling when being loaded or unloaded from a horse box, compulsive circling to the right, culminating in a seizure on day 7. Full neurologic and ocular examinations were not performed during the horse’s decline as the unpredictable nature of the horse’s movements would have made these examinations dangerous. On the basis of the proprioceptive deficits, ataxia, and circling, a forebrain lesion was suspected.

After the seizure episode on day 7, the horse was recumbent for several hours. He exhibited an intermittent nystagmus of only the right eye when lying on his left side and had loss of pupillary light reflexes. On day 8, he was unable to eat and was very uncoordinated. He continuously swayed while standing and appeared to have lost conscious proprioception in all four limbs. When asked to walk forward, the horse had to be propped up by two people and could not manage to place his feet in weight-bearing positions without assistance.

On day 9, the horse became extremely frantic and violent, repeatedly running into the walls of his stable, rearing, and falling over backward. This behavior resulted in severe self-inflicted traumatic injuries, and the horse was euthanized on humane grounds with barbiturate.

No head pressing was noted throughout the course of the disease. Pyrexia or changes in pulse or respiration were not detected. However, these parameters were not monitored during seizure and traumatic activity. No significant changes in hematology or biochemical parameters were noted in samples collected on the first and fourth days of clinical signs. Samples were not collected after the seizure episode. The case was initially diagnosed a neurologic disease of unknown origin and was sent for postmortem examination. Differential diagnoses of rabies and equine protozoal myelopathy would have been considered had the case not occurred in Ireland where these diseases are not known to occur. Equine herpes virus (EHV) 1 myeloencephalopathy was considered as it has been known to occur in the Britain; however, clinical signs were dissimilar to previously described cases, and when tested, the horse did not have a titer for EHV. The horse also tested negative for equine infectious anemia.

The horse was initially treated with 10% oxytetracycline and flunixin meglumine given IV twice daily for 4 days, and a course of Gastrogaurd (omeprazole) was recommended. On day 4, treatment was changed to a course of intramuscular penicillin and gentamicin twice daily; flunixin meglumine treatment was continued throughout the clinical course of the disease.

The horse had been resident on the premise since birth and would have had regular access to pasture. All herbaceous feed and oats were home grown. Four other horses were in training at the premise at the time of case occurrence, and mixing with horses from other training yards would have been common. The case occurred in an area with a high population density of rabbits.

2.1. Postmortem Examination

Self-inflicted injuries were numerous. The right lateral aspect of the frontal bone, the nuchal crest, the transverse lumbar processes, and the transverse processes between sacral vertebrae 3 and 4 were fractured. There was hemorrhage into the vertebral canal at the level of third cervical vertebra.

Nodules 1–3 mm in diameter were present in the dura mater over the cerebral cortices and cerebellum. Foci of malacia were present in the white matter deep to the cortex of the right cerebral hemisphere, lateral to the lateral ventricle, and caudal to the basal ganglia. The lesion was unilateral and about 1.5–2 cm in diameter.

The margo plicatus appeared ulcerated and keratinized. A serosanguinous material was present in the first meter of the small intestine, and the cecum contained a red and/or brown ingesta. The left dorsal colon had liquid contents but contained no blood.

There was right-sided congestion and edema of both lungs. The right ventricle of the heart was flaccid. The spleen was congested, consistent with euthanasia by barbiturate.

2.2. Histopathology

Multiple tissue samples were collected from the brain, spinal cord, kidney, liver, lung, myocardium, small and large intestines, skeletal muscle, and lymph nodes for histopathologic examination.

Examination of cerebral sections revealed a nonsuppurative granulomatous meningoencephalitis. Large areas of necrosis with gitter cells were present in the cerebral cortex in association with severe perivascular lymphoplasmacytic cuffing. Multinucleated giant cells and eosinophils were also present (Fig. 1). The nodules found on gross examination of the dura were focal granulomatous lesions, which included some multinucleated cells enclosed by a fibrous tissue response. Perivascular cuffing was also noted in the cerebellum. No other significant lesions were detected in the other organs examined.

Fig. 1.

Hematoxylin and eosin–stained section of the cerebral lesion found in the horse at 600× magnification. The lesion was found in the cerebral cortex caudal to the basal ganglia and lateral to the lateral ventricle in the right hemisphere.

Sections of brain lesions were stained by the ZN method. Serial sections were also stained by a standard avidin-biotin complex immunohistochemical (IHC) method applying anti-E. cuniculi antibody (rat antiserum against E. cuniculi; Medicago, Uppsala, Sweden; dilution 1:5,000). Sections stained with ZN showed the presence of small, round, intracellular and extracellular, ZN-positive structures. Intracellular structures were primarily located in macrophages and appeared in clusters in the cytosol. Extracellular structures were singular. Immunohistochemical staining showed an intracytoplasmic positive signal within scattered macrophages (Fig. 2). These structures stained basophilic on hematoxylin and eosin–stained sections.

Fig. 2.

Immunohistochemical-stained section (400×) showing positive staining in the macrophages.

2.3. Serologic Evidence of Protozoal Parasitism

Serum samples were tested for titers to E. cuniculi by Carmichael Torrance Veterinary Diagnostic Laboratory (CTVD) using an enzyme-linked immunosorbent assay (ELISA). The horse was found to have a titer of 1:320. Titers above 1:20 are considered positive to E. cuniculi.

There was no serologic evidence of exposure to Toxoplasma gondii or Neospora caninum (CTVD).

The horse was also negative for Sarcocystis neurona by Western blot (Equine Diagnostic Solutions LLC, Kentucky).

2.4. Virus Detection

A Coggins test and ELISA for equine infectious anemia were negative. Complement fixation testing for EHVs 1 and 4 was negative as was polymerase chain reaction (PCR) testing on brain tissue from the affected site. Antibodies to West Nile virus were not detected by ELISA.

2.5. PCR and Genotyping for Detection of E. cuniculi

Ten sections of formalin-fixed brain tissue (approximately 2 mm³ each) were incubated in ATL Buffer (Qiagen, Valencia, CA) at 90°C for 1 hour to partially reverse the effects of formaldehyde on nucleic acids. Each tissue section was then extracted for DNA using the QIAmp DNA Mini Kit (Qiagen). Nested PCR was performed using microsporidia specific primers (MSP) primers described by Katzwinkel-Wladarsch et al [10] that target ribosomal DNA genes of the microsporidian species reported to infect mammals. Nested PCR was performed, and the reaction mix contained 2.5 U of PuReTaq DNA polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 200 μM of each dNTP, stabilizers, and BSA using the PuReTaq Ready-To-Go Beads (Amersham; Piscataway, NJ) along with 1 μM of each primer, and 1 μL of template DNA in a final reaction final volume of 25 μL. The PCR first-round primers were 5′-TGA ATG KGT CCC TGT-3′ (MSP-1), 5′-TCA CTC GCC GCT ACT-3′ (MSP-2A), and 5′-GTT CAT TCG CAC TAC T-3′ (MSP-2B). The second-round primers were 5′-GGA ATT CAC ACC GCC CGT CRY TAT-3′ (MSP-3), 5′-CCA AGC TTA TGC TTA AGT YMAARG GGT-3′ (MSP-4A), and 5′-CCA AGC TTA TGC TTA AGT CCAGGG AG-3′ (MSP-4B). Thermal cycler conditions were 5 minutes at 95°C followed by 36 cycles of: 1 minute at 95°C, 1.5 minutes at 55°C, 3 minutes at 72°C, and then a final primer extension at 72°C for 10 minutes. Amplicons were electrophoresed in a 1% agarose gel and stained with ethidium bromide.

Amplicons of Encephalitozoon spp. are expected to be 289–305 bp and those of Enterocytozoon are 508 bp. Amplicons were detected in three of the 10 brain tissue sections that were approximately 300 bp each suggesting infection with an Encephalitozoon species. To identify the species, DNA sequencing was performed on electrophoresed amplicons that were excised from the gel and purified using the Wizard SV Gel and PCR Clean-Up System (Catalog #A9281; Promega Corporation, Madison, WI). Samples were sent to Geneway Research (Hayward, CA) for DNA sequencing. The nucleic acid sequences were subjected to BLASTn analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=WGS&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch), and the E. cuniculi genotype II was identified (Didier et al [11]) (Fig. 3).

Fig. 3.

Nested polymerase chain reaction was performed on ten tissue sections of the horse brain using MSP primers that target ribosomal DNA of microsporidia. Three of the tissue sections generated amplicons that were approximately 300 bp, suggestive for Encephalitozoon. Nucleic acid sequences of amplicons produced in lanes 1, 3, and 9 were determined to be Encephalitozoon cuniculi genotype II based on BLASTn analysis. +, positive E. cuniculi tissue culture control; −, negative tissue culture control; lanes 1–10, the ten tissue sections from this horse; M, ØX174/Hae3 marker.

3. Discussion

Encephalitozoon cuniculi is a microsporidial parasite with a wide host range. Encephalitozoon cuniculi can infect lower order animalia such as insects and fish as well as members of the higher order mammalia [1–3,5,6]. Infections by this organism can have clinical signs consistent with encephalitis, nephritis, uveitis, or abortion and/or placentitis; however, infection can remain unapparent. Fenbendazole is used to treat infection in rabbits that have nephritis or uveitis due to an E. cuniculi infection; however, the efficacy of this treatment for rabbits with the encephalitic form of the disease has not been proven.

Serologic evidence of equine infection with E. cuniculi has been demonstrated in Brazil and Israel. Interestingly, there was a strong correlation between high antibody titers and the presence of neurologic signs in Israel [5,6]. The nature and severity of these signs, however, were not documented. Encephalitozoon cuniculi has also been demonstrated in a case of equine placentitis in the United States [3] and a stillborn foal in South Africa [12].

Lesions attributable to infection tend to be granulomatous in nature with perivascular cuffing. These histopathologic features were seen in the presented case. The clustering of organisms within the macrophage seen in this case is consistent with the description of E. cuniculi parasitophorous vacuoles by Weidner [13] using electron microscopy. Granulomatous meningoencephalitis of unknown origin has previously been described in both the horse and dog [9,14].

Encephalitozoon cuniculi commonly infects rabbits, and given the exposure opportunity, the presumed source of infection was wild rabbits. However, none of the four rabbits captured in the area tested positive for serologic evidence of exposure to E. cuniculi. The incidence of E. cuniculi in laboratory rabbits varies from 5% to 75% [2]. Given that many IHC methods use rabbit antibodies as primary antibodies, caution may be required in their interpretation if E. cuniculi is a differential.

The incidence of E. cuniculi in the Irish wild rabbit population is not known. Infected rabbits are known to shed E. cuniculi spores in their urine. Ingestion, inhalation, and infection through trauma or direct contact with a mucosal membrane have been described as potential routes of infection. Vertical transmission has been described in rodents, carnivores, and nonhuman primates [1].

In the case reported here, E. cuniculi genotype II was identified. This genotype was previously identified in mice, rabbits, blue foxes, birds, and more recently in HIV-infected patients in Russia [8,15]. Other cases of equine infection with E. cuniculi have not been typed.

The results of this case report suggest that E. cuniculi should be considered in the differential diagnosis of equine encephalitis of unknown origin.