CASE IV: MW19-0525 (JPC 4152805)

 

Signalment:

6-year-old female zebra (Equus zebra)

 

History:

This zebra was born in Camp Verde, Arizona. It had a history of a dystocia in 2014 that appeared to cause neurologic problems including inability to stand and seizures that she appeared to have recovered from after 8 hours. However after the neurologic events, the zebra had some long term hind limb ataxia and seemed to have some pain after heat cycles. September 7 2017, the zebra showed signs of a head tilt, circling, pacing with front limb ataxia. The zebra was still

eating and drinking; however, the front limb ataxia progressively worsened over 3 days. September 11, 2017 the zebra was sedated for laboratory work and physical examination. The physical exam was unremarkable with dirty ears with no ticks being the primary finding. Blood was collected (see below for ante mortem bloodwork results) and dexamethasone and IV fluids were administered. The zebra had a very prolonged recovery as she was unable to stand for 6 hours post sedation. The following day, the zebra showed mild improvement. However over the course of 7 days post-sedation, the zebra had worsening clinical signs of severe ataxia, circling, and head tilt that eventually progressed to leaning on the fence and walls. There was no vaccination history. The Zebra was euthanized on September 15, 2017 and was submitted for gross and histopathological examination.

 

Gross Pathology:

On external examination, there are locally extensive, erosive to ulcerated, flat, red to dark red areas on the skin of the supraorbital region, bilaterally. The skin of the right side is more affected than the left side. The underlying subcutis of the head has multiple large areas of subcutaneous hemorrhage and edema. The brain and the spinal cord are grossly unremarkable. Internal organs are within normal limits.

 

Laboratory Results:

Complete blood count: Hct 38% (32.5-46.5%), WBC 10.3K/µL (4.3-11.4 K/µL), Neut 8.93 K/µL (2.46-7.23 K/µL), Lymph 1.082 K/µL (1.45- 5 K/µL), Monocyte 0.185 K/µL (0-0.6 K/µL), Eosinophil 0.093 K/µL (0-0.7 K/µL), Basophil 0.01 K/µL (0- 0.1 K/µL), Platelet 274 (70-250 K/µL)

Neutrophils appear slightly toxic per reviewer comments.

Fibrinogen: 400 mg/dL (100-400 mg/dL)

 

Chemistry: Glucose 55 mg/ dL (49-102 mg/dL),Creatinine 2.1 mg/dL (0.8-1.8 mg/dL), BUN 16mg/dL (11-25 mg/dL), Phosphorous 5.0 mg/ dL (2.0-4.8 mg/dL), Calcium 11.2 mg/ dL (10.2-12.8 mg/dL), Sodium 133 mmol/ L (132-141 mmol/L), Potassium 3.2 mmol/ L (2.5-5.2 mmol/L), Na:K ratio 42 (no range given), Chloride 95 mmol/L (96-106 mmol/L), TC02 (bicarbonate) 18 mmol/L (21-31mmol/L) , Anion Gap 23 mmol/L (8-18 mmol/L), Total Protein 8.1 g/dL (5.7-7.5 mg/dL), Albumin 3.2 g/dL (3.0-3.9 mg/dL), Globulin 4.9 g/dL 2.3-4.1 g/dL), AST 1,377 U/L (194-431 U/L), ALP 127 U/L (76-262 U/L), Total bilirubin 0.5 mg/dL (0.4-2.8 mg/dL), Conjugated Bilirubin 0.1 mg/dL (0.2-0.6 mg/dL), Unconjugated Bilirubin 0.4 mg/dL (0.1-2.8 mg/dL), Cholesterol 124 mg/dL (49-150 mg/dL), Creatine Kinase 873 U/L (130-497 U/L)

 

EHV-1 Real PCR Serology:

Neuropathogenic: negative

Non-neuropathogenic: negative

 

Immunohistochemistry:

Neospora caninum: Protozoal cysts exhibit strong positive immunoreactivity.

Sarcocystis neurona: Protozoal cysts exhibit negative immunoreactivity.

 

PCR:

The DNA nucleotide sequence of Apicomplexan and Neospora spp PCR confirmed that the organism is most likely Neospora caninum (performed by Michigan State University).

 

Microscopic Description:

Brain: Multifocally, the neuroparenchyma is randomly disrupted with small to large numbers of inflammatory cells, large numbers of lymphocytes, small numbers of plasma cells, few macrophages, few eosinophils, rare multinucleated giant cells and malacia. Variable degrees of perivascular cuffing up to 5 cells thick with large numbers of lymphocytes, plasma cells, macrophages, few eosinophils, and reactive vascular endothelial cells expands the Virchow Robin spaces and into surrounding the

neuroparenchyma. Astrocytes are swollen (reactive) with large amounts of cytoplasm (gemistocytes) and swollen axons are occasionally identified. Multiple large areas of clear spaces, disruption of neuroparenchyma, and vacuoles (edema) are associated with these inflammatory cells. The leptomeninges are markedly expanded with similar inflammatory infiltrates, mild hemorrhage, fibrin, necrotic debris and edema. Within these inflammatory foci, there are multiple 20-25 µm in diameter protozoal

cystic structures with a discernible outer wall containing numerous 2x4 µm oval to crescent-shaped tachyzoites and basophilic nucleus.

 

Contributor's Morphologic Diagnoses:

Meningoencephalitis, lymphoplasmacytic, histiocytic, multifocal, severe with perivascular cuffing, gliosis, free protozoal

tachyzoites and numerous protozoal cysts.

Contributor's Comment:

The current name of equine protozoal myeloencephalitis (EPM) was given at American Association of Equine Practitioners (AAEP) meeting in 1977.7 The causative agent of EPM is either Sarcocystis neurona or Neospora spp, although the majority of cases are caused by S. neurona. 3,5 Protozoa was first observed within the Toxoplasma-like encephalomyelitis in the horse in 1974 1 but reviews of these cases were attributed to S. neurona. 3 Neospora-associated EPM is uncommon but many cases have been reported.3-5,9 This is the first Neospora-associated EPM case reported in a zebra.

 

Neospora caninum was first described as a new genus and species in 1988 and caused meningoencephalomyelitis and myositis in dogs.2 Bovine neosporosis causes abortion in both dairy and beef cattle worldwide and reproductive failure leads to major economic impact. Most of the cases are aborted at 5-6 months of gestation. Neospora caninum can be transmitted transplacentally which is the major mode of transmission in cattle.2 The definitive host of N. caninum is domestic dogs which produce oocysts in feces.2 In horses, Neospora-like organisms were identified in two aborted foals and adult horses.2-4 In 1998, the new name of equine neosporosis, Neospora hughesi, is proposed.3 Neospora hughesi has been isolated and has differentiated from N. caninum by molecular and biological techniques.2,3,6 Neospora-associated EPM (N. hughesi and possibly N. caninum) in horses is an uncommon infection in United States and only one case has been reported in Canada.2,3,9

 

In this zebra case, multifocal severe lymphoplasmacytic meningoencephalitis is identified in the cerebrum, brain stem and spinal cord. Within the inflammatory foci, numerous free protozoal tachyzoites (2-4 µm) and protozoal cysts (20-30 µm in diameter) are identified. The tissue cyst wall of N. caninum is 1-4 µm thick, whereas the tissue cysts of T. gondii is less than 1 µm.3 The tissue cysts of N. hughesi can be compared with N. caninum. Tissue cysts of N. hughesi are smaller than N. caninum and the bradyzoites were smaller than those of N. caninum.3 However, it is unclear whether N. hughesi is the only Neospora species for EPM in horses.3 Immunohistochemistry (IHC) revealed a strong positive immunoreactivity for the N. caninum antibody and negative immunoreactivity to S. neurona (performed in University of Minnesota). The DNA nucleotide sequence of Apicomplexan PCR (16-1 Cap 18S ribosomal RNA gene) and N. caninum PCR (NcCalr5 NC5 marker genomic sequence) revealed 100% similarity (187/187bp and 209/209bp) to N. caninum, respectively. However, N. hughesi is closely related to N. caninum. Therefore, performed IHC and DNA sequence of N. caninum may not be differentiated between N. caninum and N. hughesi. One paper differentiated N. hughesi from N. caninum based on their immunodominant surface antigen, SAG1 and SAG1-related sequence 2 (SRS2).6 There

was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. These markers can be used to distinguish N. caninum from N. hughesi.6 Wobeser et al 2009 targeted the first internal transcribed spacer (ITS-1) region to distinguish N. hughesi from N. caninum.9

EPM is one of commonly diagnosed infectious agents in adult horses in the United States, most commonly caused by S. neurona and uncommonly by Neospora spp.2 Both N. caninum and N. hughesi have been reported and more cases of N. hughesi have been identified in recent cases.3,7,9 The definitive host of N. hughesi has not been identified and has been speculated to be small rodents or canids.7 Currently antemortem EPM is diagnosed with serum titer test from CSF and serum. All horses are believed to be susceptible to EPM, but not all infected horses with either S. neurona or N. hughesi will develop disease.7 A recent study shows the seroprevalence of N. hughesi is low in horses (between 3-10%) depending on geographic differences.7 EPM caused by Neospora spp. may be underestimated. Currently, SAG1, SRS2 and ITS-1 are pending to differentiate N. caninum and N. hughesi in this case.

 

Contributing Institution:

Diagnostic Pathology Center

Midwestern University College of Veterinary Medicine

5725 West Utopia Rd.

Glendale, AZ 85308

https://www.mwuanimalhealth.com/diagnostic-pathology-center


JPC
Diagnosis:

Cerebrum: Meningoencephalitis, necro-tizing, multifocal, severe, chronic with numerous apicomplexan cysts and extracellular zoites.

JPC Comment:

As mentioned by the contributor, additional molecular diagnostics were pending at the time of this case's submission to WSC. Subsequent molecular diagnostics utilizing the ITS-1 region revealed 100% genetic similarity to N. caninum while control N. hughesi DNA from the typed species was not detected, further supporting the diagnosis of N. caninum.8 This case is significant in that it not only represents the first reported case of N. caninum associated EPM confirmed using molecular analysis but also the first case of N. caninum associated EPM reported in a zebra. Therefore techniques utilized with this case can be applied toward retrospective and future EPM studies to better understand the prevalence and geographic distribution of N. caninum compared to N. hughesi associated EPM in equids.8

 

Neosporosis was first identified as an entity by Bjerkås et al. in Norway during the 1980s. Distributed worldwide, reported seropositivity rates for N. caninum in dogs are as high as 38% in Argentina and 22% in New Zealand. Interestingly, multiple studies have demonstrated rural canines have significantly higher seropositivity rates of compared to their urban counterparts.2

 

As previously noted, canines are the definitive host of N. caninum but can also be intermediate hosts as well. Other domestic species serving as intermediate hosts include cattle, sheep, goats, and horses. The apicomplexan parasite's lifecycle is composed of three infectious stages, with intracellular tachyzoites and cysts found in the intermediate hosts and unsporulated oocysts in definitive hosts. Cysts are typically found in the CNS but can also be found in other tissues such as placenta and liver. They have a round to oval shape and can be up to slightly more than 100 µm in diameter with an approximately 4 µm thick wall surrounding bradyzoites. In the definitive canine host, unsporulated oocysts enter the gastrointestinal tract and are passed in the feces, contaminating the environment. The oocysts become infective following sporulation and are subsequently ingested by the intermediate host.

 

Although the source of intermediate host infection of is clear and multiple intermediate hosts have been identified, canine infection in regions without exposure to known intermediate hosts suggests the existence of additional intermediate host species, such as small animals that may be preyed upon by dogs in urban areas. Infected tissues from intermediate hosts in rural regions, such as aborted fetuses, fetal membranes, and dead calves likely serve as the major sources of infection and may explain the difference in seropositivity between rural and urban canines.2

 

In addition to Neospora spp., EPM is also caused by a closely related apicomplexan parasite, Sarcocystis neurona. In both cases, equids are believed to become infected following ingestion of infective oocysts passed in the feces of the definitive host. Whereas the definitive hosts of N. caninum include the domestic dog and the coyote (Canis latrans), Sarcocystis neurona's definitive host is the opossum (Didelphis sp.). The definitive host for N. hughesi has not been identified.9

 

The moderator discussed the significance of multinucleated giant cells within the section, suggesting their presence in the CNS of any horse with a history of neurologic deficits should elevate the level of suspicion for a protozoal etiology.

 

References:

1.     Beech J, Dodd D. Toxoplasma-like encephalomyelitis in the horse. Vet Pathol. 1974;11(1):87‐96

2.     Dubey JP. Review of Neospora caninum and neosporosis in animals. Korean J Parasitol. 2003;41:1?16.

3.     Dubey JP, Lindsay DS, Saville WJ, et al. Granstrom DE, Speer CA. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet Parasitol. 2001;95(2-4):89‐131.

4.     Hamir A, Tornquist, Gerros T, et al. Neospora caninum-associated equine protozoal myeloencephalitis, Vet parasitol. 1998:79:269-274

5.     Lindsay DS, Steinberg H, Dubielzig R, et al. Central nervous system neosporosis in a foal. J Vet Diagn Invest. 1996;8:507?510.

6.     Marsh AE, Howe DK, Wang G, et al. Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2. Int J Parasitol. 1999;29(10):1575‐1582.

7.     Reed SM, Furr M, Howe DK, Johnson AL, MacKay RJ. Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention. J Vet Intern Med. 2016;30(2):491‐502.

8.     Ruppert S, Lee JK, Marsh AE. Equine Protozoal Myeloencephalitis associated with Neospora caninum in a USA captive bred zebra (Equus zebra). Vet Parasitol Reg Stud Reports. 2021;26:100620. 

9.     Wobeser BK, Godson DL, Rejmanek D, Dowling P. Equine protozoal myeloencephalitis caused by Neospora hughesi in an adult horse in Saskatchewan. Can Vet J. 2009;50(8):851‐853.

 


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