MEDICINE


sourced from two diagnostics laboratories from the authors’ institution. Owners’ consent was obtained to use muscle specimens for the study. Muscle bi- opsy specimens were collected because of suspected neuromuscular or muscle disease. The medical re- cords of the selected horses were reviewed for a definitive diagnosis. Muscle specimens were pro- cessed according to each laboratory protocol. For a comparison of findings, healthy horses were used as controls. This study was approved by an Animal Care and Use Committee.


Muscle Biopsy


Muscle biopsy specimens were snap frozen in iso- pentane precooled in liquid nitrogen and stored at 80°C until further processing. Muscle specimens were routinely processed for histological and immu- nohistochemical analysis and evaluated under light microscopy. Myofibers were characterized by fiber type (1, 2A, 2B) through ATPase reaction at prein- cubation pH of 9.8, 4.6, and 4.3. The cross-sectional area of fiber types was determined using a total of 100 muscle fibers of each type in 3 random locations, for a total of 300 myofibers of each type. Only areas without artifacts and myofibers with distinct bor- ders were measured. Immunohistochemistry to de- termine the type of inflammatory cells, if present, included clusters of differentiation (CD) for B- lymphocytes (CD20, CD79), T-lymphocytes (CD3, CD4, CD8), and macrophages (CD11c). Additional skeletal muscle specimens consisted of archived for- malin-fixed paraffin-embedded blocks and glass slides routinely stained with hematoxylin and eosin.


Molecular Analysis


To determine the sarcocyst species, DNA was ex- tracted from 15 muscle samples. Nucleic acids were extracted using the DNeasy blood and tissue kita.


Statistical Analysis


Descriptive statistics included mean, standard devi- ation, and range for all measurements. A Fisher’s exact test was used to test for an association be- tween the presence of sarcocysts as well as the num- ber of muscles with sarcocysts and state of health (diseased and healthy horses). An independent t- test was used to compare myofiber size from those with and without sarcocysts, and the number of sarcocysts per muscle specimen between horses with neuromuscular disease and controls. Statistical significance was set at P  0.05.


3. Results


Encysted parasites in skeletal muscle were identi- fied in a total of 50 equids: 35 equids with neuro- muscular disease from one of the diagnostic laboratories and 15 horses with miscellaneous dis- orders from archived formalin-fixed samples. The control group consisted of 36 horses. Skeletal mus- cle from a total of 392 equids with neuromuscular


466 2018  Vol. 64  AAEP PROCEEDINGS


disease were evaluated during the study period for a prevalence of 8.9%. Various breeds were repre- sented, including Percheron, Quarter Horse, Thor- oughbred, Arabian, Warmblood, Mustang, Paso Fino, and Icelandic pony. The mean and median age was 7 years old (range, 1–16 years of age). Diseased horses had signs compatible with multifo- cal or diffuse neuromuscular disease, such as weak- ness, short stride gait, and muscle atrophy (Fig. 1). Some horses also had muscle stiffness, fascicula- tions, and apparent pain upon palpation. Altered gait had an undetermined etiology, and ataxia was seen in 2 horses. Horses with dysphagia had swol- len and apparently painful tongues. Of the 36 con- trol horses, only 1 had a single encysted parasite for a prevalence of 2.7% within this control population. Twenty of 35 horses had a complete blood cell


count and serum biochemical profile done. Normo- cytic normochromic anemia (packed cell volume, 28–30%; reference range, 32–45%) was identified in 3 of 20 horses and eosinophilia in 5 of 20 (2.5–4.7% of total white blood cells). An elevation of muscle enzymes was observed in 13 of 20 horses: creatine kinase ranged 560–250,000 IU/L (reference range, 119–287 IU/L) and aspartate aminotransferase ranged 999–29,568 IU/L (reference range, 168–494 IU/L).


Although equine protozoal myeloencephalopathy


(EPM) was not suspected in these horses based on neuroanatomical localization of neuromuscular dis- ease, an immunofluorescent antibody test for S. neu- rona and Neospora hughesi antibodies was performed in 17 horses. Of these, 6 horses were negative for antibodies, 9 had low titers or below the cut-off value provided by the diagnostic laboratory at the authors’ institution, and 2 had titers above the cut-off value considered supportive of EPM. Two additional horses with clinically suspected EPM based on pro- gressive multifocal asymmetrical central nervous dis- ease had serum and CSF titers supportive of EPM (horse 1, S. neurona; horse 2, S. neurona, N. hughesi). Fifty-one (83.6%) of 61 muscles from 35 equids


with neuromuscular disease had encysted parasites, versus 1 (1.4%) of 72 muscles from 36 healthy horses (significant difference, P  0.001). Based on histo- logical findings, the horses were grouped into dis- ease categories: noninflammatory myopathies (n 18/35 horses), inflammatory myopathies (n  4/35), neurogenic muscle atrophy (n  2/35), and normal histology (n1/35). Noninflammatory myopathies included nonexertional rhabdomyolysis of undeter- mined cause, nutritional myodegeneration due to vitamin E and selenium deficiency, polysaccharide storage myopathy, vacuolar myopathy of undeter- mined cause, pituitary pars intermedia dysfunction, and malignant hyperthermia. Inflammatory my- opathy consisted of myositis with a predominance of CD8 T-lymphocytes with marked myonecrosis (Fig. 2). Myofibers containing sarcocysts were sig- nificantly larger than ones without parasites. Mo-


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