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Sarcocystis fayeri in skeletal muscle of horses with neuromuscular disease
Accepted Manuscript Title: Sarcocystis fayeri in skeletal muscle of equids with neuromuscular disease Author: Monica Aleman, Karen Shapiro, Silvia Sisó, Diane C. Williams, Daniel Rejmanek, Beatriz Aguilar, Patricia A. Conrad PII: DOI: Reference:
S0960-8966(15)00741-5 http://dx.doi.org/doi:10.1016/j.nmd.2015.09.014 NMD 3102
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
21-7-2015 7-9-2015 22-9-2015
Please cite this article as: Monica Aleman, Karen Shapiro, Silvia Sisó, Diane C. Williams, Daniel Rejmanek, Beatriz Aguilar, Patricia A. Conrad, Sarcocystis fayeri in skeletal muscle of equids with neuromuscular disease, Neuromuscular Disorders (2015), http://dx.doi.org/doi:10.1016/j.nmd.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Sarcocystis fayeri in skeletal muscle of equids with neuromuscular disease
Monica Alemana*, Karen Shapirob, Silvia Sisób, Diane C. Williamsc, Daniel Rejmanekb, Beatriz Aguilarb, Patricia A. Conradb
a
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis-California, United States
of America b
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis-
California, United States of America c
The William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis-
California, United States of America
*Corresponding author: Monica Aleman, MVZ Cert., PhD, Dipl. ACVIM (Internal Medicine, Neurology); SVM: Department of Medicine and Epidemiology, Tupper Hall 2108, One Shields Avenue, University of California, Davis-California, United States of America, Tel: +(530)-752-1363, [email protected]
Abbreviations:
NDL
Neuromuscular Disease Laboratory
Authors declare:
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2 No conflicts of interest The study was performed at the University of California at Davis The study was supported by the Hart research grant and the Neuromuscular Disease Laboratory, University of California, Davis
Highlights:
Sarcocystis fayeri infection was common in equids with neuromuscular disease
Sarcocystosis could be associated with myopathic and neurogenic processes
Similar to humans sarcocystosis, cardiomyopathy can also occur in horses
Assuming Sarcocysts spp. always being an incidental finding might be inaccurate
Abstract (200 words) Recent reports of Sarcocystis fayeri-induced toxicity in people consuming horse meat warrant investigation on the prevalence and molecular characterization of Sarcocystis spp. infection in equids. Sarcocysts in skeletal muscle of equids has been commonly regarded as an incidental finding. In this study, we investigated the prevalence of sarcocysts in skeletal muscle of equids with
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3 neuromuscular disease. Our findings indicated that Sarcocystis fayeri infection was common in young mature equids with neuromuscular disease and could be associated with myopathic and neurogenic processes. The number of infected muscles and number of sarcocysts per muscle were significantly higher in diseased than in control horses. Sarcocystis fayeri was predominantly found in low oxidative highly glycolytic myofibers. This pathogen had a high glycolytic metabolism. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait deficits, and dysphagia in horses with involvement of the tongue and esophagus. Horses with myositis were lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. Although this study did not establish causality but a possible association (8.9% of cases) with disease; the assumption of Sarcocysts spp. being an incidental finding in every case might be inaccurate.
Key words:
Equine, muscle, parasites, protozoa
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4 1. Introduction Reports of parasitic infections in humans caused by consumption of horse meat include trichinellosis and sarcocystosis.[1, 2] However, the vast majority of reports to date have targeted trichinellosis.[1, 3] Outbreaks of food poisoning due to S. fayeri as the result of ingesting raw horse meat have not been described until recently.[2, 4] Humans are the definitive host for S. hominis and S. suihominis; whereas cattle and swine are the intermediate hosts.[5, 6] These sarcocysts have been found in skeletal and cardiac muscle, larynx, pharynx, and upper esophagus in humans.[5] Intestinal sarcocystosis in people has resulted from eating raw or undercooked beef and pork containing mature sarcocysts of S. hominis and S. suihominis, respectively.[6] Humans can also become accidental intermediate hosts for other Sarcocystis species by ingesting food or drinking water contaminated with fecal sporocysts from an infected definitive host.[6] Subsequently, muscle sarcocystosis in people occurs via hematogenous dissemination.[6, 7]
Parasitic infestation of skeletal muscle in equine species includes nematodes and protozoans.[1, 8] Nematodes infestation comprises Trichinella spiralis, Trichinella britovi, and Haycocknema perplexum.[9-13] Intracellular protozoans include Sarcocystis bertrami, S. equicanis, S. fayeri, S. asinus, S. neurona, and Trypanosoma evansi.[8, 14-16] Similar to other species, the presence of sarcocysts in equine skeletal muscle has been considered an incidental finding since muscle pathology has not been observed.[17] However, there have been sporadic reports of myositis in horses associated with Sarcocystis spp.[18, 19] Furthermore, experimental infection of ponies and horses with S. fayeri has resulted in severe myositis and stiff gait.[20] The prevalence of sarcocysts in skeletal muscle of horses has been reported to range from 4 to 93%.[8, 21] However, comprehensive medical information and full neurologic
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5 examination are lacking in those studies. Therefore, state of health or disease was not determined. Prevalence of infection might vary with horse’s age, muscle sampled, and environmental exposure.[16] An association between parasite load and age (higher load if horse > 8 to 10 years old) was noted in 3 studies.[16, 21] Diaphragm, heart, tongue, and esophageal muscles have been reported to have higher parasite loads.[16, 21] The prevalence of intramyofiber protozoal cysts in a postmortem study of 229 equids was 8% (18 horses, 1 pony)[17], and was the third most common histological finding in skeletal muscle.[17]
Identification of intramyofiber-encysted parasites in horses has been possible through histological, ultrastructural, immunohistochemical, and molecular studies.[14, 22, 23] The prevalence of intramyofiber encysted protozoans has not been investigated specifically in horses with neuromuscular disease. Therefore, the purpose of this study was to determine the prevalence of encysted parasites in skeletal muscle of equids with neuromuscular disease, molecularly characterize the species, describe histochemical features, and report association with myopathic and neuropathic disorders.
2. Materials and Methods 2.1. Animals 2.1.1. Diseased equids This study included equine skeletal muscle with evidence of encysted parasites within myofibers sourced from the Neuromuscular Disease Laboratory (NDL) and the hospital anatomopathological archive from the University of California at Davis from the years of
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6 2000 to 2014. Owners’ consent was obtained to use muscle specimens for the study. Muscle biopsies from the NDL were collected because of suspected neuromuscular or muscle disease. The medical records of the selected horses were reviewed for a definitive diagnosis. Muscle specimens were processed according to each laboratory protocols.
2.1.2. Controls Archived biopsies from the gluteus medius and semimembranosus muscles from 36 clinically healthy horses with a total of 72 muscle specimens were included in the study. State of health was determined through a complete physical and neurologic examination by one of the authors (MA), and blood work within reference ranges (complete blood cell count and chemistry panel). This study was approved by the UC Davis an Animal Care and Use Committee.
2.2. Muscle biopsy 2.2.1. Histopathology and immunohistochemistry (NDL) Equine muscle biopsy specimens received at the NDL were snap frozen in isopentane pre-cooled in liquid nitrogen and stored at 80°C until further processing. Muscle specimens from the NDL were routinely processed for histological and immunohistochemical analysis and evaluated under light microscopy as described.[24] In brief, the following histochemical stains and reactions were performed: hematoxylin and eosin, modified Gomori trichrome, periodic acid Schiff, phosphorylase, esterase, Staphylococcal protein A-horseradish peroxidase, myosin ATPase at preincubation pH of 9.8, 4.6, and 4.3, nicotinamide adenine dinucleotide, succinate
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7 dehydrogenase, acid phosphatase, alkaline phosphatase, and oil red O. Immunohistochemistry to determine the type of inflammatory cells if present included clusters of differentiation for B-lymphocytes (CD20, CD79), T-lymphocytes (CD3, CD4, CD8), and macrophages (CD11c).
2.2.2. Morphologic and morphometric evaluation (NDL) Myofibers were characterized by fiber type (1, 2A, 2B) through ATPase reaction at preincubation pH of 9.8, 4.6, and 4.3. Myofibers were also characterized by shape, size, number per high power field (HPF), and number of nuclei per myofiber. Light microscopic images in transverse planes were evaluated with NIH Image™ software (NIH Image, Bethesda, MA, USA). Cross-sectional area (CSA) 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 borders were measured. Coefficients of variability (CV) of muscle fiber types were calculated as published elsewhere.[24] The data collected for myofibers containing parasites included fiber type, CSA, CV, and presence of necrosis or inflammatory cells.
2.3. Postmortem muscle (Pathology) Skeletal muscle specimens from the UC Davis Department of Comparative Pathology consisted of archived formalin-fixed, paraffin embedded blocks, and glass slides routinely stained with hematoxylin and eosin. To retrieve such samples, the equine data base of our institution was searched using the key words muscle, parasites, encysted, protozoa, and sarcocysts.
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8
2.4. Molecular analysis Fifteen muscle samples (n = 14 snap frozen, n = 1 paraffin embedded) were used for DNA extraction. Nucleic acids were extracted using the DNeasy blood and tissue kit (Qiagen). The extracted specimen was incubated with 180 μl ATL buffer and 30 μl proteinase K and incubated at 56C overnight. The remainder of the extraction process was carried out following manufacturer’s instructions.
For Sarcocystis DNA amplification via polymerase chain reaction (PCR), a nested primer set targeting the 18S rRNA gene was designed.[25] Primer sequences were described in detail elsewhere[25], and included the S5 and S4 as external, and S7 and S2 as internal primers. Reaction mixtures (50 l total) included 5 l of 10X PCR buffer containing 15 mM MgCl2, 1 l of 10 mM dNTP mixture, 25 pmol forward primer, 25 pmol reverse primer, 1.5 U Taq polymerase, 2 l DNA template for external reactions and 1 l DNA for internal reactions. Amplification conditions were as follows: initial denaturation for 3 min at 94C, followed by 35 cycles of 40 sec at 95C, annealing for 40 sec at 58C (external) or 59C (internal), elongation for 90 sec at 72C, and post PCR extension for 4 min at 72C, followed by holding at 4C. In all PCR assays, tissue culture-derived S. neurona DNA was used as a positive control, and muscle samples from healthy horses as well as reagent blanks used as negative controls.
Amplified DNA products were run on gels (2% agarose) using electrophoresis and visualized using ethidium bromide under ultraviolet illumination. Samples that yielded DNA amplification were purified (Qiagen QIA quick Gel Extraction Kit) and submitted for
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9 sequencing (DNA sequencing facility, UC Davis, CA). For sequence analysis, the forward and reverse DNA sequences were aligned using a multiple sequence comparison by log-expectation (MUSCLE) method via Geneious software (Biomatters, Auckland, New Zealand), ends trimmed, and the consensus sequence compared with Genbank reference sequences for S. fayeri using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To obtain cleaner sequences for positive specimens, the amplified DNA product from 6 horses was further cloned into a PCR 4 Topo vector using the Topo TA Cloning kit and following manufacturer instructions (Life Technologies Inc., Burlington, ON). Cloned products were subsequently purified and submitted for sequencing as described above.
2.5. Statistical analysis Descriptive statistics included mean, standard deviation (SD), and range for all measurements. A Fisher’s exact test was used to test for an association between presence of sarcocysts as well as the number 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 number of sarcocysts per muscle specimen between horses with neuromuscular disease and controls. Statistical significance was set at < 0.05.
3. Results 3.1. Animals Encysted parasites in skeletal muscle were identified in a total of 50 equids: 35 equids with neuromuscular disease from the NDL, and 15 horses with miscellaneous disorders from the Comparative Pathology laboratory. The control group consisted of 36 horses.
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10 3.1.1. Diseased equids Encysted parasites were identified within myofibers of skeletal muscle from 35 (n = 34 horses, n = 1 mule) of 392 equids with neuromuscular disease submitted to the NDL during the study period for a prevalence of 8.9%. The breed distribution of 34 horses with neuromuscular disease consisted of Percheron (n = 10), Quarter horse and related breeds (n = 9), Thoroughbred (n = 4), Arabian (n = 3), Warmblood (n = 3), Mustang (n = 3), Paso Fino (n = 1), and Icelandic (n =1). Sex distribution comprised 18 females and 17 males (castrated 14, intact 3). Mean and median age was 7 years old with a range of 1 to 16 years of age. The geographic location of these horses included California (n = 29), Colorado (n = 1), Idaho (n = 1), Indiana (n = 1), Ohio (n = 1), Oregon (n = 1), and the United Kingdom (n = 1).
Nineteen of the 35 equids were also examined by one of the authors (MA) to determine physical and neurologic status. Diseased horses had signs compatible with multifocal or diffuse neuromuscular disease such as muscle atrophy, weakness, and reduced muscle tone. Eleven of 19 horses had muscle stiffness, short stride gait, and apparent muscle pain upon palpation. Altered gait of undetermined cause was observed in 4 of 19 horses, and 2 of 19 horses had ataxia. Two of 19 horses had swollen and apparently painful tongues in addition to muscle atrophy, stiffness, weakness, and altered gait. Normocytic normochromic anemia (PCV 28-30%, reference range 32-45%) was identified in 3 of 20 horses that had a CBC done. Eosinophilia was found in 5 of 20 horses (>1% of total WBC; 2.5-4.7%), and elevation of muscle enzymes was observed in 13 of 20 horses. In these 13 horses, creatine kinase ranged from 560 to 250,000 IU/L (reference range 119-287 IU/L) and aspartate aminotransferase from 999 to 29,568 IU/L (reference range 168-
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11 494 IU/L). Although equine protozoal myeloencephalopathy was not suspected based on a neuroanatomical localization of neuromuscular disease in these horses except for 2 horses (progressive multifocal asymmetrical central nervous disease); an immunofluorescent antibody test for S. neurona and N. hughesi antibodies was done using serum and cerebrospinal fluid from 15 of 19 horses. This test was negative (n = 6/15) or below the cut-off titer (n = 5/15) to support probability of clinical EPM in 11 horses but above the cut-off titer in 4 horses (N. hughesi [2 equids: horse, mule]; S. neurona [1 horse]; and S. neurona and N. hughesi) [1 horse both]). Two of these 4 equids with a titer above cut-off value were suspected to have EPM based on neurologic examination.
3.1.2. Controls Of the 36 control horses, only 1 had a single encysted parasite in the semimembranosus muscle for a prevalence of 2.7% within this group population. Control horses consisted of Thoroughbred (n = 16), Quarter Horse (n = 11), Standardbred (n = 4), Arabian (n = 3), Warmblood (n = 1), and Percheron (n = 1) breeds. There were 23 females and 13 castrated males. Mean and median age was 13 years old with a range of 2 to 28 years of age. There was no statistical difference (P = 0.3) when comparing the number of individuals with sarcocysts in diseased versus healthy horses.
3.2. Muscle biopsy 3.2.1. Muscle histopathology and immunohistochemistry (NDL)
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12 Fifty-one (83.6%) of 61 muscles from 35 equids with neuromuscular disease, versus one (1.4%) of 72 muscles from 36 healthy horses had encysted parasites (Table 1). This difference was statistically significant (P < 0.001). Skeletal muscle containing cysts included semimembranosus (n = 21 of 25 muscles), gluteus medius (n = 13/15), sacrocaudalis dorsalis lateralis (n = 7/8), triceps brachii (n = 5/7), biceps brachii (n = 1/2), longissimus lumbaris (n = 1/1), extensor digitalis lateralis (n = 1/1), cranial tibialis (n = 1/1), and genioglossus (n = 1/1) muscles. The number of parasites observed ranged from 2 to 21 per muscle in diseased horses versus a single parasite in a single muscle in one control horse. This difference was statistically significant (P < 0.001). A single sarcocyst per myofiber was seen for all muscle specimens except for one myofiber that had 2 sarcocysts. All muscles were composed of types 1, 2A, and 2B myofibers. The sacrocaudalis dorsalis lateralis was composed of 25-40% of type 1 and the rest of type 2 myofibers; whereas all other muscles studied had a lower proportion of type 1 myofibers ranging from 15 to 30%. This myofiber type distribution was not different to the available NDL database from muscle of healthy horses. Therefore, myofiber type distribution did not appear to be disrupted by the presence of sarcocysts. Myofiber type distribution of encysted parasites based on myosin ATPase reaction at various preincubation pHs (9.8, 4.6, and 4.3) consisted of 7.3% in type 1, 14.7% in 2A, and 78% in 2B myofibers (Table 1, Figure 1). Based on succinate dehydrogenase reaction, encysted parasites were seen predominantly in low oxidative fibers (90%) (Table 1, Figure 2). However, sarcocysts had moderate to high oxidative activity (Figure 2). The location of the myofibers containing sarcocysts was predominantly adjacent to connective tissue where blood vessels were visualized (not shown). Based on periodic acid Schiff, sarcocysts had high glycolytic activity. Sarcoplasmic masses were visualized in muscles of 13 horses.
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13 Based on muscle histological findings, these horses were grouped under disease categories such as non-inflammatory myopathies (n = 18/35 horses), inflammatory myopathies (n = 4/35), neurogenic muscle atrophy (n = 10/35), mixed (inflammatory myopathy and neurogenic muscle atrophy, n = 2/35), and normal (n = 1/35) (Table 2, Figure 3). Non-inflammatory myopathies included nonexertional rhabdomyolysis (n = 11/18 horses) of undetermined cause, nutritional myodegeneration due to selenium and vitamin E deficiency (low concentrations in blood and muscle, n = 3/18), polysaccharide storage myopathy (PSSM, n = 2/18), vacuolar myopathy of undetermined cause (n = 1/18), and pituitary pars intermedia dysfunction myopathy (n = 1/18). Genetic testing for malignant hyperthermia (MH RyR1 mutation) and PSSM (GYS1 mutation) was performed in horses with rhabdomyolysis and identified in 2 more horses with PSSM type 1. The Quarter horse with vacuolar myopathy tested positive for hyperkalemic periodic paralysis (HYPP SCN4A mutation). Inflammatory myopathies in 6 horses were characterized by inflammatory cell infiltrates (CD3+: predominantly CD8+, Figure 3A), marked myonecrosis, and elevation of muscle enzymes (creatine kinase and aspartate aminotransferase). Sacrocaudalis dorsalis lateralis muscles from one horse and one mule had histological findings (neurogenic muscle atrophy of both fiber types [1, 2] but more profound on type 1 myofibers) suggestive of equine motor neuron disease.
3.2.2. Morphologic and morphometric evaluation (NDL) The myofibers containing an encysted parasite had an oval to round shape. Neighboring myofibers varied from normal (polygonal) to atrophic (small: anguloid or angular) morphology. The mean CSA of the myofibers containing the encysted parasite was 6.2 x 103 µ2 and ranged from 5.4 to 9.2 x 103 µ2 and were statistically larger than the CSA of myofibers not containing cysts when specific
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14 myofiber types were compared. The coefficient of variability of affected muscle fiber types was over 30% and considered higher than that from healthy muscle.[24] On longitudinal sections, sarcocysts were visualized extending the length of the myofiber (not shown).
3.3. Postmortem muscle (Pathology) Twenty-six muscle specimens from 15 horses had encysted parasites in skeletal (n = 22) and cardiac (n = 4) muscles. Estimation of the prevalence of infection based upon postmortem evaluation was not possible since muscle histologic examination was not routinely done for every case. All horses were native to California. These 15 horses were euthanized for various reasons such as gastrointestinal (n = 3; colon torsion, lipoma, colonic adenocarcinoma), cardiac (n = 3: cardiomyopathy with heart failure), neurological (n = 1: cervical vertebral malformation), muscle (n = 5: myositis), hepatic (n = 2: amyloidosis, pyrrolizidine alkaloid toxicosis), and renal (n = 1: tubular nephrosis) disorders. Horses diagnosed with cardiomyopathy had signs compatible with heart failure such as tachycardia, anasarca, jugular pulses, tachypnea, respiratory distress, and enlarged heart based on echocardiogram examination (performed in one case). The breed distribution included Quarter Horses (n = 4), Thoroughbred (n = 3), Arabian (n = 2), Standardbred (n = 2), and one of each: Percheron, Mustang, Welsh pony, and American Miniature horse. There were 10 females and 5 males (castrated 3, intact 2). Horses’ age ranged from 3 months to 20 years old (mean 9.2, median 8 years of age). Affected skeletal muscle included the semimembranosus (n = 6), genioglossus (n = 3), unknown (n = 7), and one each muscle splenius cervicalis, gluteus medius, triceps brachii, diaphragm, laryngeal, and esophageal muscles (this horse had dysphagia). Muscle pathology was observed in 66% (n = 10/15) horses as follows: non-inflammatory (n = 1/15, Figure 4A), inflammatory myopathy (myositis, n = 4/15), neurogenic muscle atrophy
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15 and myositis (n = 1/15, Figure 4B), and cardiomyopathy (n = 4/15, 3 had heart failure) (Table 2). The remaining 5 horses had no histological abnormalities in skeletal muscle other than the presence of the parasite. Sarcocyst morphology was easier to visualize on formalin-fixed tissue, and distinction between metrocytes and bradyzoites was more apparent (Figure 4B). Sarcocysts wall thickness and villar protrusions were variable. However, only thick walled sarcocysts were observed. Samples for electron microscopy were not available to describe morphology in more detail.
3.4. Molecular analysis Of 15 sarcocyst-positive muscle samples that were submitted for molecular testing, 6 yielded DNA amplification with sequences that aligned with S. fayeri. Due to the large number of ambiguous and mixed bases, 3 cloned products for each positive sample were further analyzed via sequence analysis. All cloned sequences aligned with S. fayeri sequences previously deposited in GenBank, but were highly polymorphic both between horses as well as within cloned products from the same horse samples (Table 3).
4. Discussion This study showed an apparently higher prevalence of equids with neuromuscular disease and concomitant sarcocysts in skeletal muscle (8.9%) than that of a control healthy population (2.7%); though this was not statistically significant. However, the prevalence of skeletal muscles with sarcocysts was significantly higher in equids with neuromuscular disease (83.6%, n = 51/61 muscles) than in healthy horses (1.4%, n = 1/72 muscles). Furthermore, the number of parasites per muscle sample was significantly higher in equids
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16 with neuromuscular disease (ranging from 2 to 21 per muscle examined) than healthy horses (1 total from a single muscle from one horse). Muscle wasting, weakness, with or without apparent muscle pain, and gait deficits were commonly observed abnormalities. Horses with dysphagia (n = 6/48) had involvement of the tongue (n = 5) or esophagus (n = 1). Myofibers containing a sarcocyst were significantly larger than those without the parasite which is similar to the description by others.[8] However, in the study by Gunn there was no identification of Sarcocysts species.[8] Sarcocystis fayeri was more commonly found in low oxidative highly glycolytic fibers which might reflect the metabolic demands of the parasite. However, it appears that S. fayeri has a high oxidative metabolism based on the presence of intracellular high succinate dehydrogenase activity. Sequence analysis revealed the presence of S. fayeri DNA in 6 of 15 muscle samples that had sarcocysts visualized microscopically, with a high degree of polymorphisms present in cloned sequences.
Sarcocystis is classified in the phylum Apicomplexa, along with Eimeria, Toxoplasma, and Cystoisospora.[26] Sarcocystis species are ubiquitous in nature and found worldwide. Sarcocysts have been reported to be highly prevalent in musculature of horses.[8, 16, 21] Species identified in the muscle of equids include S. asinus, S. bertrami, S. equicanis, and S. fayeri;[8, 15, 16, 27] with only one report of a single filly having S. neurona.[14] These species utilize canids as definitive hosts except for S. neurona which uses the opossum.[28] Horses in America can be infected with 2 Sarcocystis species; S. fayeri and S. neurona.[29] Although, it has been reported that the presence of sarcocysts in skeletal muscle is an incidental finding; this assumption has not been fully investigated due to lack of comprehensive clinical studies with only 3 sporadic cases of severe myositis in association with the presence of Sarcocysts
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17 spp.[18, 19] Two of these 3 horses had granulomatous and eosinophilic myositis on which S. fayeri was found intralesionally and their signs included chronic weight loss, abnormal gait, apparent muscle pain, and dysphagia.[19] Further, there have been 3 isolated cases with chronic progressive debilitating disease of undetermined cause described as weight loss, weakness, and lethargy on which S. fayeri was found in skeletal muscle.[15, 30, 31] Additionally, one of these cases had difficulty eating and swallowing and had ataxia or altered gait.[29] The results of the present study do not support S. fayeri as always being an incidental bystander. However, evidence for a causative relationship between S. fayeri and neuromuscular disease is still lacking. Further, horses from this study had concurrent myopathies that could have led to the observed clinical signs. However, the signs described here were similar to those isolated cases with Sarcocystis spp. described in the literature.[15, 29-31] In comparison, healthy horses had normal musculature and no clinical signs of disease including the single case with a sarcocyst. Gunn and Fraher found sarcocysts in muscle of 3 of 74 horses with no signs of disease.[8] Also, of interest from this study was the finding of 4 horses with a cardiomyopathy and concurrent sarcocysts in cardiac muscle, of which 3 resulted in heart failure and euthanasia. The 4th horse was euthanized due to gastrointestinal disease and the cardiomyopathy was identified on postmortem evaluation. Sarcocysts’ role in the pathogenesis of a cardiomyopathy in these horses could not be entirely excluded. Further, sarcocystosis affecting cardiac muscle resulting in cardiomyopathy has been described in human medicine.[32]
There was no breed predilection, except for the Percheron breed that appeared to be overrepresented (20%) when compared to the Percheron population from our hospital. However, the majority of these horses (8 of 10) came from a single farm. Therefore, it is
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18 possible that this overrepresentation might have depicted a local problem or high seroprevalence of antibodies for S. neurona and N. hughesi in this farm and not a true overrepresentation of breed. Saville et al reported that horses with S. fayeri infection might be misdiagnosed as being infected with S. neurona based on IFAT.[20] However, in the present study only 2 horses had serum IFA titers above the cut-off value considered supportive of probability of clinical disease.[33, 34]
Although there was no apparent age predilection in this study, diseased equids appeared to be mainly young mature horses (median 7, mean 7.9 + 5.6 years) with 83.3% being younger than 15 years of age. This is in contrast to reports of older animals having higher parasite loads.[16, 21] The single healthy horse with a sarcocyst in this study was 15 years old. It is noteworthy that specimens with high parasite loads such as the diaphragm, heart, tongue, and esophageal muscles were not routinely evaluated because the majority of the muscle samples collected in this study were biopsied from live animals.[20] Sex predilection has not been reported, and was not observed in this study. Prevalence of infection could vary with geographic location throughout the world.[16, 17, 21] In this study, geographic origin of equids with neuromuscular disease included California, Colorado, Indiana, Ohio, Oregon, and the United Kingdom. The majority of the equids with neuromuscular disease in this study (82.9%) and all healthy horses were natives of California.
Of all 50 diseased equids, 54% (n = 27/50), 22% (n = 11/50), and 6% (3/50) had a myopathic, neuropathic, and mixed process based on muscle histological analysis, respectively. Evidence of cardiomyopathy was seen on 8% (n = 4/50). Of 27 horses with a myopathic
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19 process, 19 had a non-inflammatory myopathy, and 8 had an inflammatory myopathy (myositis presumed associated with the presence of sarcocysts). Additionally, 3 horses had myositis and neurogenic muscle atrophy. CD8+ T-lymphocytes were the predominant cell population in skeletal muscle with evidence of myositis. Studies in mice support a critical role of CD8+ cells in the prevention of infection by S. neurona.[35] Two other studies reported myositis associated with the presence of sarcocysts.[18, 19] Horses with myositis in the present study had severe polyphasic histological lesions and marked cellular infiltration of myocytes and perimysium. These horses were lethargic, apparently painful, stiff, and reluctant to move. Two of these horses also had a swollen tongue and were dysphagic. These findings were different to those described in Quarter horses with suspected immune mediated myositis on which a cause was not identified and cellular infiltrates consisted mainly of CD4+ T-lymphocytes.[36] In humans with muscular sarcocystosis; acute and long-term persistent or intermittent myalgia, myositis, muscle wasting, arthralgia, fatigue, headache, cough from bronchospasm, rashes, facial swelling, and fever are some common manifestations.[26]
As horse meat is consumed by people worldwide, evaluating the distribution and identity of parasites in horse muscles is relevant for securing public health.[2, 4, 37] Health status of horses going to slaughter facilities is unknown, posing a risk for human disease. Humans could be either definitive or intermediate hosts to specific species of sarcocysts.[26] Sarcocystis infection has caused both, intestinal and muscular sarcocystosis in humans.[2, 26] Other forms such as cardiomyopathy and glomerulonephritis have also been reported.[26, 32] Sarcocystosis can occur by the ingestion of contaminated undercooked or raw meat, water, or fresh produce.[26] Recent outbreaks of food poisoning in humans caused by the ingestion of raw horse meat contaminated with S. fayeri have been
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20 described in Japan.[2, 4] In that study, a 15-kDa protein was extracted from horse meat containing S. fayeri.[2] This protein produced a positive enterotoxic response in rabbits characterized by diarrhea and death.[2]
Molecular analysis of the cloned parasite DNA isolated from horse muscle samples in this study were closely aligned (up to 100% identity) with sequences with GenBank samples derived from a horse meat inspection facility in Japan, where S. fayeri-caused meat poisoning has been reported.[2, 4] The high degree of polymorphisms obtained between cloned samples is likely due to variability within the high number of repetitive 18S rRNA genomic DNA copies. However, mixed infection with different S. fayeri strains is also possible. Due to the recent connection between S. fayeri-contaminated horse meat and a threat to human health, further molecular research should investigate whether toxicity from this parasite is associated with specific S. fayeri strains. Targeted meat screening for presence of toxigenic S. fayeri could provide a valuable approach for ensuring safe distribution of horse meat prior to ii reaching human consumer.
5. Conclusion Sarcocystis fayeri infection is common in young mature equids with neuromuscular disease. Sarcocystis fayeri has a moderate to high oxidative and glycolytic metabolism but has preferential establishment in low oxidative highly glycolytic myofibers of skeletal muscle. Sarcocystis fayeri infection can be associated with myopathic (non-inflammatory, inflammatory [myositis]), neuropathic, or both processes. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait
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21 deficits and in some cases dysphagia if involving the tongue and/or esophagus. Horses with myositis can be lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. Although causality between S. fayeri and neuromuscular disease has not been definitively established, the assumption of Sarcocysts spp. being an incidental finding may not be accurate for all cases. Further, as the health status of horses entering slaughter facilities may be unknown, ingestion of horse meat containing S. fayeri can pose a health hazard to human consumers.
6. References [1] Boireau P, Vallee I, Roman T, Perret C, Mingyuan L, Gamble HR, et al. Trichinella in horses: a low frequency infection with high human risk. Vet Parasitol 2000;93:309-20. [2] Kamata Y, Saito M, Irikura D, Yahata Y, Ohnishi T, Bessho T, et al. A toxin isolated from Sarcocystis fayeri in raw horsemeat may be responsible for food poisoning. J Food Prot 2014;77:814-9. [3] Jimenez-Cardoso E, Caballero-Garcia ML, Uribe-Gutierrez G, Trejo-Hernandez E, Gay-Jimenez FR. Frequency of Trichinella spiralis in blood and muscles of horses from two slaughter houses (industrial and rural) in the State of Mexico, Mexico. Vet Mex 2005;36:269-78. [4] Harada S, Furukawa M, Tokuoka E, Matsumoto K, Yahiro S, Miyasaka J, et al. Control of toxicity of Sarcocystis fayeri in horsemeat by freezing treatment and prevention of food poisoning. Shokuhin Eiseigaku Zasshi 2013;54:198-203. [5] Lele VR, Dhopavkar PV, Kher A. Sarcocysts infection in man. Indian J Pathol Microbiol 1986;29:87-90.
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22 [6] Fayer R. Sarcocystis spp. in human infections. Clin Microbiol Rev 2004;17:894-902. [7] Italiano CM, Wong KT, AbuBakar S, Lau YL, Ramli N, Syed Omar SF, et al. Sarcocystis nesbitti causes acute, relapsing febrile myositis with a high attack rate: description of a large outbreak of muscular sarcocystosis in Pangkor Island, Malaysia, 2012. PLoS Negl Trop Dix 2014;8:e2876:1-9. [8] Gunn HM, Fraher JP. Incidence of sarcocysts in skeletal muscles of horses. Vet Parasitol 1992;42:33-40. [9] Eckert J, Ossent P. Haycocknema-like nematodes in muscle fibres of a horse. Vet Parasitol 2006;139:256-61. [10] Pozio E, Celano GV, Sacchi L, Pavia C, Rossi P, Tamburrini A, et al. Distribution of Trichinella spiralis larvae in muscles from a naturally infected horse. Vet Parasitol 1998;74:19-27. [11] Pozio E, Paterlini F, Pedarra C, Sacchi L, Bugarini R, Goffredo E, et al. Predilection sites of Trichinella spiralis larvae in naturally infected horses. J Helminthol 1999;73:233-7. [12] Arriaga C, Yepez-Mulia L, Viveros N, Adame LA, Zarlenga DS, Lichtenfels JR, et al. Detection of Trichinella spiralis muscle larvae in naturally infected horses. J Parasitol 1995;81:781-3. [13] Liciardi M, Marucci G, Addis G, Ludovisi A, Gomez Morales MA, Deiana B, et al. Trichinella britovi and Trichinella spiralis mixed infection in a horse from Poland. Vet Parasitol 2009;161:345-8. [14] Mullaney T, Murphy AJ, Kiupel M, Bell JA, Rossano MG, Mansfield LS. Evidence to support horses as intermediate hosts for Sarcocystis neurona. Vet Parasitol 2005;133:27-36.
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23 [15] Cawthorn RJ, Clark M, Hudson R, Friesen D. Histological and ultrastructural appearance of severe Sarcocystis fayeri infection in a malnourished horse. J Vet Diagn Invest 1990;2:342-5. [16] Edwards GT. Prevalence of equine sarcocystis in British horses and a comparison of two detection methods. Vet Rec 1984;115:265-7. [17] Valentine BA. Pathologic findings in equine muscle (excluding polysaccharide storage): a necropsy study. J Vet Diagn Invest 2008;20:572-9. [18] Traub-Dargatz JL, Schlipf JW, Jr., Granstrom DE, Ingram JT, Shelton GD, Getzy DM, et al. Multifocal myositis associated with Sarcocystis sp in a horse. J Am Vet Med Assoc 1994;205:1574-6. [19] Herd HR, Sula MM, Starkey LA, Panciera RJ, Johnson EM, Snider TA, et al. Sarcocysts fayeri-induced granulomatous and eosinophilic myositis in 2 related horses. Vet Pathol 2015;DOI:10.1177/0300985815584073:1-4. [20] Saville WJA, Dubey JP, Oglesbee MJ, Sofaly CD, Marsh AE, Elitsur E, et al. Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses. J Parasitol 2004;90:1487-91. [21] Fukuyo M, Battsetseg G, Byambaa B. Prevalence of Sarcocystis infection in horses in Mongolia. Southeast Asian J Trop Med Public Health 2002;33:718-9. [22] Dubey JP, Lindsay DS, Fritz D, Speer CA. Structure of Sarcocystis neurona sarcocysts. J Parasitol 2001;87:1323-7. [23] Lindsay DS, Mitchell SM, Vianna MC, Dubey JP. Sarcocystis neurona (protozoa: apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development. J Parasitol 2004;90:461-5.
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24 [24] Aleman M, Watson JL, Williams DC, LeCouteur RA, Nieto JE, Shelton GD. Myopathy in horses with pituitary pars intermedia dysfunction (Cushing's disease). Neuromuscul Disord 2006;16:737-44. [25] Fischer S, Odening K. Characterization of bovine Sarcocystis species by analysis of their 18S ribosomal DNA sequences. J Parasitol 1998;84:50-4. [26] Fayer R, Esposito DH, Dubey JP. Human infections with Sarcocystis species. Clin Microbiol Rev 2015;28:295-311. [27] Hilali M, Nassar AM. Ultrastructure of Sarcocystis spp. from donkeys (Equus asinus) in Egypt. Vet Parasitol 1987;23:179-83. [28] Dubey JP, Lindsay DS, Saville WJ, Reed SM, Granstrom DE, Speer CA. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet Parasitol 2001;95:89-131. [29] Dubey JP, Streitel RH, Stromberg PC, Toussant MJ. Sarcocystis fayeri sp. n. from the horse. J Parasitol 1977;63:443-7. [30] Tinling SP, Cardinet GH, 3rd, Blythe LL, Cohen M, Vonderfecht SL. A light and electron microscopic study of sarcocysts in a horse. J Parasitol 1980;66:458-65. [31] Fayer R, Hounsel C, Giles RC. Chronic illness in a Sarcocystis-infected pony. Vet Rec 1983;113:216-7. [32] Beaver PC, Gadgil RK, Morera P. Sarcoystis in man: a review and report of five cases. Am J Trop Med Hyg 1979;28:819-44. [33] Duarte PC, Daft BM, Conrad PA, Packham AE, Gardner IA. Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis. J Vet Diagn Invest 2003;15:8-13.
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25 [34] Duarte PC, Daft BM, Conrad PA, Packham AE, Saville WJ, MacKay RJ, et al. Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses. J Parasitol 2004;90:379-86. [35] Witonsky SG, Gogal RM, Duncan RB, Norton H, Ward D, Lindsay DS. Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells. Int J Parasitol 2005;35:113-23. [36] Lewis SS, Valberg SJ, Nielsen IL. Suspected immune-mediated myositis in horses. J Vet Intern Med 2007;21:495-503. [37] Belaunzaran X, Bessa RJB, Lavin P, Mantecon AR, Kramer JKG, Aldai N. Horse-meat for human consumption-current research and future opportunities. Meat Sci 2015;108:74-81.
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26 Figures Figure 1. Sarcocyst within a myofiber of fresh frozen equine skeletal muscle on myosin ATPase reaction at various preincubation pH. (1A) Reaction at pH 9.8. Note sarcocyst within type 2 myofiber (dark color), bar 100µm. (1B) Reaction at pH 9.8. Note sarcocyst within type 1 myofiber (light, pink), bar 50 µm.
Figure 2. Succinate dehydrogenase reaction on fresh frozen equine muscle tissue. (2A) Note sarcocyst within a low oxidative myofiber (light color), bar 100 µm. (2B) Note sarcocyst within a highly oxidative myofiber (darker color) surrounded by low oxidative fibers (lighter color), bar 50 µm.
Figure 3. Disease groups based on skeletal muscle histological findings. (3A) Inflammatory myopathy. Immunohistochemistry depicting CD8+ cell infiltration, bar 100 µm. (3B) Neurogenic muscle atrophy. Note several myofibers with angular atrophy (*), bar 100 µm.
Figure 4. Formalin-fixed skeletal muscle on hematoxylin and eosin. (4A) Non-inflammatory rhabdomyolysis. Note enlarged oval myofiber containing a single sarcocyst with metrocytes (arrow head) and bradyzoites (arrow) within cyst (bar 20 µm). (4B) Myositis with combined neurogenic muscle atrophy (bar 50 µm).
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27
Table Table 1. Sarcocysts in skeletal muscle. This table depicts total muscle samples from 35 horses with neuromuscular disease (NMD, N = 61 muscles), and 36 healthy horses (Control, N = 72 muscles). Total number of myofibers containing sarcocysts is shown according to myofiber type distribution (1, 2A, 2B; high versus low oxidative) for horses with neuromuscular disease and controls. Muscle sarcocysts = number of muscles with sarcocysts, SM = semimembranosus, GLUT = gluteus medius, SCDL = sacrocaudalis dorsalis lateralis, TRI = triceps brachii, BICEPS = biceps brachii, LONG = longissimus lumbaris, EXT DL = extensor digitalis lateralis, Cr TIB = cranial tibialis, GENIO = genioglossus.
MYOFIBERS containing sarcocysts (N = 181)
NMD
MUSCLE
MUSCLE (N = 61) SM (N = 24) GLUT (N = 13) SCDL (N = 7)
Sarcocysts (N = 51/61)
1
2A
2B
High oxidative
Low oxidative
21/25
6
10
25
8
33
13/15
2
4
32
3
21
7/8
3
4
40
3
27
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28 TRI (N = 7) BICEPS (N = 2) LONG (N = 1) EXT DL (N = 1) Cr TIB (N = 1) GENIO (N = 1)
5/7
0
2
10
1
11
1/2
0
2
8
0
10
1/1
0
0
8
0
8
1/1
0
0
9
0
9
1/1
0
0
6
0
6
1/1
0
0
10
0
10
TOTAL
11 (6%)
22 (12.2%)
148 (81.7%)
27 (14.9%)
154 (85.1%)
CONTROL MUSCLE MUSCLE Sarcocysts (N = 72) (N = 1/72) SM 1/36 (N = 36) GLUT 0/36 (N = 36) TOTAL
MYOFIBERS containing sarcocysts (N = 1) 1
2A
2B
High oxydative
Low oxydative
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
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29 Table 2. Disease groups based on muscle histological findings of horses with neuromuscular disease (NMD, N = 35), and postmortem cases (PATH, N = 15). NIM = non-inflammatory myopathy, IM = inflammatory myopathy, NE = neurogenic muscle atrophy, MIX (inflammatory myopathy + neurogenic muscle atrophy), CARDIO = cardiomyopathy, NORMAL = no histological abnormalities, NA = not applicable, 1* = horse with concurrent cardiomyopathy.
DISEASED HORSES (N = 50)
NMD (N = 35)
PATH (N = 15)
TOTAL (N = 50)
NIM IM NE MIX (IM + NE) CARDIO NORMAL
18 4 10 2 NA 1
1* 4 1 1 4 5
19 (1*) 8 11 3 4 6
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30 Table 3. Percent identity between clones of Sarcocystis fayeri 18S rRNA gene products obtained from horse skeletal muscle. The heat map coloring corresponds to paired sequences that are less (light pink) to more (dark pink) similar to each other.
HORSE 1
HORSE 2
HORSE 3
HORSE 4
HORSE 5
HORSE 6
CLONE 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
1
HORSE 1 2
3
92.2 88.5 81.6 81.9 81.6 81.7 81.6 81.4 81.4 81.6 81.4 81.4 81.2 81.7 81.6 81.7 82.1
90.0 85.7 85.7 85.7 85.9 85.7 85.9 85.6 85.7 85.6 85.6 85.4 83.4 85.7 85.9 82.9
81.5 81.8 81.5 81.6 81.5 80.7 81.5 81.5 81.5 81.5 81.1 80.4 81.5 81.6 80.3
1
HORSE 2 2
3
99.2 99.7 99.8 99.7 98.8 99.5 99.7 99.5 99.5 99.3 94.8 99.7 99.8 94.4
99.2 99.3 99.2 98.3 99.0 99.2 99.0 99.0 98.8 95.1 99.2 99.3 94.7
99.8 99.7 98.8 99.5 99.7 99.5 99.5 99.3 94.8 99.7 99.8 94.4
1
HORSE 3 2
3
98.8 99.0 99.7 99.8 99.7 99.7 99.5 94.9 99.8 100.0 94.5
98.8 99.8 100.0 99.8 99.5 99.7 94.8 99.7 99.8 94.4
98.5 98.8 98.5 98.6 98.5 94.6 98.8 99.0 94.2
1
HORSE 4 2
3
1
99.8 100.0 99.3 99.5 94.6 99.5 99.7 94.2
99.8 99.5 99.7 94.8 99.7 99.8 94.4
99.3 99.5 94.6 99.5 99.7 94.2
99.1 94.6 99.5 99.7 94.4
H
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31
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S0960-8966(15)00741-5 http://dx.doi.org/doi:10.1016/j.nmd.2015.09.014 NMD 3102
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
21-7-2015 7-9-2015 22-9-2015
Please cite this article as: Monica Aleman, Karen Shapiro, Silvia Sisó, Diane C. Williams, Daniel Rejmanek, Beatriz Aguilar, Patricia A. Conrad, Sarcocystis fayeri in skeletal muscle of equids with neuromuscular disease, Neuromuscular Disorders (2015), http://dx.doi.org/doi:10.1016/j.nmd.2015.09.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Sarcocystis fayeri in skeletal muscle of equids with neuromuscular disease
Monica Alemana*, Karen Shapirob, Silvia Sisób, Diane C. Williamsc, Daniel Rejmanekb, Beatriz Aguilarb, Patricia A. Conradb
a
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis-California, United States
of America b
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis-
California, United States of America c
The William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis-
California, United States of America
*Corresponding author: Monica Aleman, MVZ Cert., PhD, Dipl. ACVIM (Internal Medicine, Neurology); SVM: Department of Medicine and Epidemiology, Tupper Hall 2108, One Shields Avenue, University of California, Davis-California, United States of America, Tel: +(530)-752-1363, [email protected]
Abbreviations:
NDL
Neuromuscular Disease Laboratory
Authors declare:
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2 No conflicts of interest The study was performed at the University of California at Davis The study was supported by the Hart research grant and the Neuromuscular Disease Laboratory, University of California, Davis
Highlights:
Sarcocystis fayeri infection was common in equids with neuromuscular disease
Sarcocystosis could be associated with myopathic and neurogenic processes
Similar to humans sarcocystosis, cardiomyopathy can also occur in horses
Assuming Sarcocysts spp. always being an incidental finding might be inaccurate
Abstract (200 words) Recent reports of Sarcocystis fayeri-induced toxicity in people consuming horse meat warrant investigation on the prevalence and molecular characterization of Sarcocystis spp. infection in equids. Sarcocysts in skeletal muscle of equids has been commonly regarded as an incidental finding. In this study, we investigated the prevalence of sarcocysts in skeletal muscle of equids with
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3 neuromuscular disease. Our findings indicated that Sarcocystis fayeri infection was common in young mature equids with neuromuscular disease and could be associated with myopathic and neurogenic processes. The number of infected muscles and number of sarcocysts per muscle were significantly higher in diseased than in control horses. Sarcocystis fayeri was predominantly found in low oxidative highly glycolytic myofibers. This pathogen had a high glycolytic metabolism. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait deficits, and dysphagia in horses with involvement of the tongue and esophagus. Horses with myositis were lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. Although this study did not establish causality but a possible association (8.9% of cases) with disease; the assumption of Sarcocysts spp. being an incidental finding in every case might be inaccurate.
Key words:
Equine, muscle, parasites, protozoa
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4 1. Introduction Reports of parasitic infections in humans caused by consumption of horse meat include trichinellosis and sarcocystosis.[1, 2] However, the vast majority of reports to date have targeted trichinellosis.[1, 3] Outbreaks of food poisoning due to S. fayeri as the result of ingesting raw horse meat have not been described until recently.[2, 4] Humans are the definitive host for S. hominis and S. suihominis; whereas cattle and swine are the intermediate hosts.[5, 6] These sarcocysts have been found in skeletal and cardiac muscle, larynx, pharynx, and upper esophagus in humans.[5] Intestinal sarcocystosis in people has resulted from eating raw or undercooked beef and pork containing mature sarcocysts of S. hominis and S. suihominis, respectively.[6] Humans can also become accidental intermediate hosts for other Sarcocystis species by ingesting food or drinking water contaminated with fecal sporocysts from an infected definitive host.[6] Subsequently, muscle sarcocystosis in people occurs via hematogenous dissemination.[6, 7]
Parasitic infestation of skeletal muscle in equine species includes nematodes and protozoans.[1, 8] Nematodes infestation comprises Trichinella spiralis, Trichinella britovi, and Haycocknema perplexum.[9-13] Intracellular protozoans include Sarcocystis bertrami, S. equicanis, S. fayeri, S. asinus, S. neurona, and Trypanosoma evansi.[8, 14-16] Similar to other species, the presence of sarcocysts in equine skeletal muscle has been considered an incidental finding since muscle pathology has not been observed.[17] However, there have been sporadic reports of myositis in horses associated with Sarcocystis spp.[18, 19] Furthermore, experimental infection of ponies and horses with S. fayeri has resulted in severe myositis and stiff gait.[20] The prevalence of sarcocysts in skeletal muscle of horses has been reported to range from 4 to 93%.[8, 21] However, comprehensive medical information and full neurologic
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5 examination are lacking in those studies. Therefore, state of health or disease was not determined. Prevalence of infection might vary with horse’s age, muscle sampled, and environmental exposure.[16] An association between parasite load and age (higher load if horse > 8 to 10 years old) was noted in 3 studies.[16, 21] Diaphragm, heart, tongue, and esophageal muscles have been reported to have higher parasite loads.[16, 21] The prevalence of intramyofiber protozoal cysts in a postmortem study of 229 equids was 8% (18 horses, 1 pony)[17], and was the third most common histological finding in skeletal muscle.[17]
Identification of intramyofiber-encysted parasites in horses has been possible through histological, ultrastructural, immunohistochemical, and molecular studies.[14, 22, 23] The prevalence of intramyofiber encysted protozoans has not been investigated specifically in horses with neuromuscular disease. Therefore, the purpose of this study was to determine the prevalence of encysted parasites in skeletal muscle of equids with neuromuscular disease, molecularly characterize the species, describe histochemical features, and report association with myopathic and neuropathic disorders.
2. Materials and Methods 2.1. Animals 2.1.1. Diseased equids This study included equine skeletal muscle with evidence of encysted parasites within myofibers sourced from the Neuromuscular Disease Laboratory (NDL) and the hospital anatomopathological archive from the University of California at Davis from the years of
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6 2000 to 2014. Owners’ consent was obtained to use muscle specimens for the study. Muscle biopsies from the NDL were collected because of suspected neuromuscular or muscle disease. The medical records of the selected horses were reviewed for a definitive diagnosis. Muscle specimens were processed according to each laboratory protocols.
2.1.2. Controls Archived biopsies from the gluteus medius and semimembranosus muscles from 36 clinically healthy horses with a total of 72 muscle specimens were included in the study. State of health was determined through a complete physical and neurologic examination by one of the authors (MA), and blood work within reference ranges (complete blood cell count and chemistry panel). This study was approved by the UC Davis an Animal Care and Use Committee.
2.2. Muscle biopsy 2.2.1. Histopathology and immunohistochemistry (NDL) Equine muscle biopsy specimens received at the NDL were snap frozen in isopentane pre-cooled in liquid nitrogen and stored at 80°C until further processing. Muscle specimens from the NDL were routinely processed for histological and immunohistochemical analysis and evaluated under light microscopy as described.[24] In brief, the following histochemical stains and reactions were performed: hematoxylin and eosin, modified Gomori trichrome, periodic acid Schiff, phosphorylase, esterase, Staphylococcal protein A-horseradish peroxidase, myosin ATPase at preincubation pH of 9.8, 4.6, and 4.3, nicotinamide adenine dinucleotide, succinate
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7 dehydrogenase, acid phosphatase, alkaline phosphatase, and oil red O. Immunohistochemistry to determine the type of inflammatory cells if present included clusters of differentiation for B-lymphocytes (CD20, CD79), T-lymphocytes (CD3, CD4, CD8), and macrophages (CD11c).
2.2.2. Morphologic and morphometric evaluation (NDL) Myofibers were characterized by fiber type (1, 2A, 2B) through ATPase reaction at preincubation pH of 9.8, 4.6, and 4.3. Myofibers were also characterized by shape, size, number per high power field (HPF), and number of nuclei per myofiber. Light microscopic images in transverse planes were evaluated with NIH Image™ software (NIH Image, Bethesda, MA, USA). Cross-sectional area (CSA) 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 borders were measured. Coefficients of variability (CV) of muscle fiber types were calculated as published elsewhere.[24] The data collected for myofibers containing parasites included fiber type, CSA, CV, and presence of necrosis or inflammatory cells.
2.3. Postmortem muscle (Pathology) Skeletal muscle specimens from the UC Davis Department of Comparative Pathology consisted of archived formalin-fixed, paraffin embedded blocks, and glass slides routinely stained with hematoxylin and eosin. To retrieve such samples, the equine data base of our institution was searched using the key words muscle, parasites, encysted, protozoa, and sarcocysts.
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8
2.4. Molecular analysis Fifteen muscle samples (n = 14 snap frozen, n = 1 paraffin embedded) were used for DNA extraction. Nucleic acids were extracted using the DNeasy blood and tissue kit (Qiagen). The extracted specimen was incubated with 180 μl ATL buffer and 30 μl proteinase K and incubated at 56C overnight. The remainder of the extraction process was carried out following manufacturer’s instructions.
For Sarcocystis DNA amplification via polymerase chain reaction (PCR), a nested primer set targeting the 18S rRNA gene was designed.[25] Primer sequences were described in detail elsewhere[25], and included the S5 and S4 as external, and S7 and S2 as internal primers. Reaction mixtures (50 l total) included 5 l of 10X PCR buffer containing 15 mM MgCl2, 1 l of 10 mM dNTP mixture, 25 pmol forward primer, 25 pmol reverse primer, 1.5 U Taq polymerase, 2 l DNA template for external reactions and 1 l DNA for internal reactions. Amplification conditions were as follows: initial denaturation for 3 min at 94C, followed by 35 cycles of 40 sec at 95C, annealing for 40 sec at 58C (external) or 59C (internal), elongation for 90 sec at 72C, and post PCR extension for 4 min at 72C, followed by holding at 4C. In all PCR assays, tissue culture-derived S. neurona DNA was used as a positive control, and muscle samples from healthy horses as well as reagent blanks used as negative controls.
Amplified DNA products were run on gels (2% agarose) using electrophoresis and visualized using ethidium bromide under ultraviolet illumination. Samples that yielded DNA amplification were purified (Qiagen QIA quick Gel Extraction Kit) and submitted for
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9 sequencing (DNA sequencing facility, UC Davis, CA). For sequence analysis, the forward and reverse DNA sequences were aligned using a multiple sequence comparison by log-expectation (MUSCLE) method via Geneious software (Biomatters, Auckland, New Zealand), ends trimmed, and the consensus sequence compared with Genbank reference sequences for S. fayeri using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To obtain cleaner sequences for positive specimens, the amplified DNA product from 6 horses was further cloned into a PCR 4 Topo vector using the Topo TA Cloning kit and following manufacturer instructions (Life Technologies Inc., Burlington, ON). Cloned products were subsequently purified and submitted for sequencing as described above.
2.5. Statistical analysis Descriptive statistics included mean, standard deviation (SD), and range for all measurements. A Fisher’s exact test was used to test for an association between presence of sarcocysts as well as the number 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 number of sarcocysts per muscle specimen between horses with neuromuscular disease and controls. Statistical significance was set at < 0.05.
3. Results 3.1. Animals Encysted parasites in skeletal muscle were identified in a total of 50 equids: 35 equids with neuromuscular disease from the NDL, and 15 horses with miscellaneous disorders from the Comparative Pathology laboratory. The control group consisted of 36 horses.
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10 3.1.1. Diseased equids Encysted parasites were identified within myofibers of skeletal muscle from 35 (n = 34 horses, n = 1 mule) of 392 equids with neuromuscular disease submitted to the NDL during the study period for a prevalence of 8.9%. The breed distribution of 34 horses with neuromuscular disease consisted of Percheron (n = 10), Quarter horse and related breeds (n = 9), Thoroughbred (n = 4), Arabian (n = 3), Warmblood (n = 3), Mustang (n = 3), Paso Fino (n = 1), and Icelandic (n =1). Sex distribution comprised 18 females and 17 males (castrated 14, intact 3). Mean and median age was 7 years old with a range of 1 to 16 years of age. The geographic location of these horses included California (n = 29), Colorado (n = 1), Idaho (n = 1), Indiana (n = 1), Ohio (n = 1), Oregon (n = 1), and the United Kingdom (n = 1).
Nineteen of the 35 equids were also examined by one of the authors (MA) to determine physical and neurologic status. Diseased horses had signs compatible with multifocal or diffuse neuromuscular disease such as muscle atrophy, weakness, and reduced muscle tone. Eleven of 19 horses had muscle stiffness, short stride gait, and apparent muscle pain upon palpation. Altered gait of undetermined cause was observed in 4 of 19 horses, and 2 of 19 horses had ataxia. Two of 19 horses had swollen and apparently painful tongues in addition to muscle atrophy, stiffness, weakness, and altered gait. Normocytic normochromic anemia (PCV 28-30%, reference range 32-45%) was identified in 3 of 20 horses that had a CBC done. Eosinophilia was found in 5 of 20 horses (>1% of total WBC; 2.5-4.7%), and elevation of muscle enzymes was observed in 13 of 20 horses. In these 13 horses, creatine kinase ranged from 560 to 250,000 IU/L (reference range 119-287 IU/L) and aspartate aminotransferase from 999 to 29,568 IU/L (reference range 168-
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11 494 IU/L). Although equine protozoal myeloencephalopathy was not suspected based on a neuroanatomical localization of neuromuscular disease in these horses except for 2 horses (progressive multifocal asymmetrical central nervous disease); an immunofluorescent antibody test for S. neurona and N. hughesi antibodies was done using serum and cerebrospinal fluid from 15 of 19 horses. This test was negative (n = 6/15) or below the cut-off titer (n = 5/15) to support probability of clinical EPM in 11 horses but above the cut-off titer in 4 horses (N. hughesi [2 equids: horse, mule]; S. neurona [1 horse]; and S. neurona and N. hughesi) [1 horse both]). Two of these 4 equids with a titer above cut-off value were suspected to have EPM based on neurologic examination.
3.1.2. Controls Of the 36 control horses, only 1 had a single encysted parasite in the semimembranosus muscle for a prevalence of 2.7% within this group population. Control horses consisted of Thoroughbred (n = 16), Quarter Horse (n = 11), Standardbred (n = 4), Arabian (n = 3), Warmblood (n = 1), and Percheron (n = 1) breeds. There were 23 females and 13 castrated males. Mean and median age was 13 years old with a range of 2 to 28 years of age. There was no statistical difference (P = 0.3) when comparing the number of individuals with sarcocysts in diseased versus healthy horses.
3.2. Muscle biopsy 3.2.1. Muscle histopathology and immunohistochemistry (NDL)
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12 Fifty-one (83.6%) of 61 muscles from 35 equids with neuromuscular disease, versus one (1.4%) of 72 muscles from 36 healthy horses had encysted parasites (Table 1). This difference was statistically significant (P < 0.001). Skeletal muscle containing cysts included semimembranosus (n = 21 of 25 muscles), gluteus medius (n = 13/15), sacrocaudalis dorsalis lateralis (n = 7/8), triceps brachii (n = 5/7), biceps brachii (n = 1/2), longissimus lumbaris (n = 1/1), extensor digitalis lateralis (n = 1/1), cranial tibialis (n = 1/1), and genioglossus (n = 1/1) muscles. The number of parasites observed ranged from 2 to 21 per muscle in diseased horses versus a single parasite in a single muscle in one control horse. This difference was statistically significant (P < 0.001). A single sarcocyst per myofiber was seen for all muscle specimens except for one myofiber that had 2 sarcocysts. All muscles were composed of types 1, 2A, and 2B myofibers. The sacrocaudalis dorsalis lateralis was composed of 25-40% of type 1 and the rest of type 2 myofibers; whereas all other muscles studied had a lower proportion of type 1 myofibers ranging from 15 to 30%. This myofiber type distribution was not different to the available NDL database from muscle of healthy horses. Therefore, myofiber type distribution did not appear to be disrupted by the presence of sarcocysts. Myofiber type distribution of encysted parasites based on myosin ATPase reaction at various preincubation pHs (9.8, 4.6, and 4.3) consisted of 7.3% in type 1, 14.7% in 2A, and 78% in 2B myofibers (Table 1, Figure 1). Based on succinate dehydrogenase reaction, encysted parasites were seen predominantly in low oxidative fibers (90%) (Table 1, Figure 2). However, sarcocysts had moderate to high oxidative activity (Figure 2). The location of the myofibers containing sarcocysts was predominantly adjacent to connective tissue where blood vessels were visualized (not shown). Based on periodic acid Schiff, sarcocysts had high glycolytic activity. Sarcoplasmic masses were visualized in muscles of 13 horses.
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13 Based on muscle histological findings, these horses were grouped under disease categories such as non-inflammatory myopathies (n = 18/35 horses), inflammatory myopathies (n = 4/35), neurogenic muscle atrophy (n = 10/35), mixed (inflammatory myopathy and neurogenic muscle atrophy, n = 2/35), and normal (n = 1/35) (Table 2, Figure 3). Non-inflammatory myopathies included nonexertional rhabdomyolysis (n = 11/18 horses) of undetermined cause, nutritional myodegeneration due to selenium and vitamin E deficiency (low concentrations in blood and muscle, n = 3/18), polysaccharide storage myopathy (PSSM, n = 2/18), vacuolar myopathy of undetermined cause (n = 1/18), and pituitary pars intermedia dysfunction myopathy (n = 1/18). Genetic testing for malignant hyperthermia (MH RyR1 mutation) and PSSM (GYS1 mutation) was performed in horses with rhabdomyolysis and identified in 2 more horses with PSSM type 1. The Quarter horse with vacuolar myopathy tested positive for hyperkalemic periodic paralysis (HYPP SCN4A mutation). Inflammatory myopathies in 6 horses were characterized by inflammatory cell infiltrates (CD3+: predominantly CD8+, Figure 3A), marked myonecrosis, and elevation of muscle enzymes (creatine kinase and aspartate aminotransferase). Sacrocaudalis dorsalis lateralis muscles from one horse and one mule had histological findings (neurogenic muscle atrophy of both fiber types [1, 2] but more profound on type 1 myofibers) suggestive of equine motor neuron disease.
3.2.2. Morphologic and morphometric evaluation (NDL) The myofibers containing an encysted parasite had an oval to round shape. Neighboring myofibers varied from normal (polygonal) to atrophic (small: anguloid or angular) morphology. The mean CSA of the myofibers containing the encysted parasite was 6.2 x 103 µ2 and ranged from 5.4 to 9.2 x 103 µ2 and were statistically larger than the CSA of myofibers not containing cysts when specific
Page 13 of 31
14 myofiber types were compared. The coefficient of variability of affected muscle fiber types was over 30% and considered higher than that from healthy muscle.[24] On longitudinal sections, sarcocysts were visualized extending the length of the myofiber (not shown).
3.3. Postmortem muscle (Pathology) Twenty-six muscle specimens from 15 horses had encysted parasites in skeletal (n = 22) and cardiac (n = 4) muscles. Estimation of the prevalence of infection based upon postmortem evaluation was not possible since muscle histologic examination was not routinely done for every case. All horses were native to California. These 15 horses were euthanized for various reasons such as gastrointestinal (n = 3; colon torsion, lipoma, colonic adenocarcinoma), cardiac (n = 3: cardiomyopathy with heart failure), neurological (n = 1: cervical vertebral malformation), muscle (n = 5: myositis), hepatic (n = 2: amyloidosis, pyrrolizidine alkaloid toxicosis), and renal (n = 1: tubular nephrosis) disorders. Horses diagnosed with cardiomyopathy had signs compatible with heart failure such as tachycardia, anasarca, jugular pulses, tachypnea, respiratory distress, and enlarged heart based on echocardiogram examination (performed in one case). The breed distribution included Quarter Horses (n = 4), Thoroughbred (n = 3), Arabian (n = 2), Standardbred (n = 2), and one of each: Percheron, Mustang, Welsh pony, and American Miniature horse. There were 10 females and 5 males (castrated 3, intact 2). Horses’ age ranged from 3 months to 20 years old (mean 9.2, median 8 years of age). Affected skeletal muscle included the semimembranosus (n = 6), genioglossus (n = 3), unknown (n = 7), and one each muscle splenius cervicalis, gluteus medius, triceps brachii, diaphragm, laryngeal, and esophageal muscles (this horse had dysphagia). Muscle pathology was observed in 66% (n = 10/15) horses as follows: non-inflammatory (n = 1/15, Figure 4A), inflammatory myopathy (myositis, n = 4/15), neurogenic muscle atrophy
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15 and myositis (n = 1/15, Figure 4B), and cardiomyopathy (n = 4/15, 3 had heart failure) (Table 2). The remaining 5 horses had no histological abnormalities in skeletal muscle other than the presence of the parasite. Sarcocyst morphology was easier to visualize on formalin-fixed tissue, and distinction between metrocytes and bradyzoites was more apparent (Figure 4B). Sarcocysts wall thickness and villar protrusions were variable. However, only thick walled sarcocysts were observed. Samples for electron microscopy were not available to describe morphology in more detail.
3.4. Molecular analysis Of 15 sarcocyst-positive muscle samples that were submitted for molecular testing, 6 yielded DNA amplification with sequences that aligned with S. fayeri. Due to the large number of ambiguous and mixed bases, 3 cloned products for each positive sample were further analyzed via sequence analysis. All cloned sequences aligned with S. fayeri sequences previously deposited in GenBank, but were highly polymorphic both between horses as well as within cloned products from the same horse samples (Table 3).
4. Discussion This study showed an apparently higher prevalence of equids with neuromuscular disease and concomitant sarcocysts in skeletal muscle (8.9%) than that of a control healthy population (2.7%); though this was not statistically significant. However, the prevalence of skeletal muscles with sarcocysts was significantly higher in equids with neuromuscular disease (83.6%, n = 51/61 muscles) than in healthy horses (1.4%, n = 1/72 muscles). Furthermore, the number of parasites per muscle sample was significantly higher in equids
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16 with neuromuscular disease (ranging from 2 to 21 per muscle examined) than healthy horses (1 total from a single muscle from one horse). Muscle wasting, weakness, with or without apparent muscle pain, and gait deficits were commonly observed abnormalities. Horses with dysphagia (n = 6/48) had involvement of the tongue (n = 5) or esophagus (n = 1). Myofibers containing a sarcocyst were significantly larger than those without the parasite which is similar to the description by others.[8] However, in the study by Gunn there was no identification of Sarcocysts species.[8] Sarcocystis fayeri was more commonly found in low oxidative highly glycolytic fibers which might reflect the metabolic demands of the parasite. However, it appears that S. fayeri has a high oxidative metabolism based on the presence of intracellular high succinate dehydrogenase activity. Sequence analysis revealed the presence of S. fayeri DNA in 6 of 15 muscle samples that had sarcocysts visualized microscopically, with a high degree of polymorphisms present in cloned sequences.
Sarcocystis is classified in the phylum Apicomplexa, along with Eimeria, Toxoplasma, and Cystoisospora.[26] Sarcocystis species are ubiquitous in nature and found worldwide. Sarcocysts have been reported to be highly prevalent in musculature of horses.[8, 16, 21] Species identified in the muscle of equids include S. asinus, S. bertrami, S. equicanis, and S. fayeri;[8, 15, 16, 27] with only one report of a single filly having S. neurona.[14] These species utilize canids as definitive hosts except for S. neurona which uses the opossum.[28] Horses in America can be infected with 2 Sarcocystis species; S. fayeri and S. neurona.[29] Although, it has been reported that the presence of sarcocysts in skeletal muscle is an incidental finding; this assumption has not been fully investigated due to lack of comprehensive clinical studies with only 3 sporadic cases of severe myositis in association with the presence of Sarcocysts
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17 spp.[18, 19] Two of these 3 horses had granulomatous and eosinophilic myositis on which S. fayeri was found intralesionally and their signs included chronic weight loss, abnormal gait, apparent muscle pain, and dysphagia.[19] Further, there have been 3 isolated cases with chronic progressive debilitating disease of undetermined cause described as weight loss, weakness, and lethargy on which S. fayeri was found in skeletal muscle.[15, 30, 31] Additionally, one of these cases had difficulty eating and swallowing and had ataxia or altered gait.[29] The results of the present study do not support S. fayeri as always being an incidental bystander. However, evidence for a causative relationship between S. fayeri and neuromuscular disease is still lacking. Further, horses from this study had concurrent myopathies that could have led to the observed clinical signs. However, the signs described here were similar to those isolated cases with Sarcocystis spp. described in the literature.[15, 29-31] In comparison, healthy horses had normal musculature and no clinical signs of disease including the single case with a sarcocyst. Gunn and Fraher found sarcocysts in muscle of 3 of 74 horses with no signs of disease.[8] Also, of interest from this study was the finding of 4 horses with a cardiomyopathy and concurrent sarcocysts in cardiac muscle, of which 3 resulted in heart failure and euthanasia. The 4th horse was euthanized due to gastrointestinal disease and the cardiomyopathy was identified on postmortem evaluation. Sarcocysts’ role in the pathogenesis of a cardiomyopathy in these horses could not be entirely excluded. Further, sarcocystosis affecting cardiac muscle resulting in cardiomyopathy has been described in human medicine.[32]
There was no breed predilection, except for the Percheron breed that appeared to be overrepresented (20%) when compared to the Percheron population from our hospital. However, the majority of these horses (8 of 10) came from a single farm. Therefore, it is
Page 17 of 31
18 possible that this overrepresentation might have depicted a local problem or high seroprevalence of antibodies for S. neurona and N. hughesi in this farm and not a true overrepresentation of breed. Saville et al reported that horses with S. fayeri infection might be misdiagnosed as being infected with S. neurona based on IFAT.[20] However, in the present study only 2 horses had serum IFA titers above the cut-off value considered supportive of probability of clinical disease.[33, 34]
Although there was no apparent age predilection in this study, diseased equids appeared to be mainly young mature horses (median 7, mean 7.9 + 5.6 years) with 83.3% being younger than 15 years of age. This is in contrast to reports of older animals having higher parasite loads.[16, 21] The single healthy horse with a sarcocyst in this study was 15 years old. It is noteworthy that specimens with high parasite loads such as the diaphragm, heart, tongue, and esophageal muscles were not routinely evaluated because the majority of the muscle samples collected in this study were biopsied from live animals.[20] Sex predilection has not been reported, and was not observed in this study. Prevalence of infection could vary with geographic location throughout the world.[16, 17, 21] In this study, geographic origin of equids with neuromuscular disease included California, Colorado, Indiana, Ohio, Oregon, and the United Kingdom. The majority of the equids with neuromuscular disease in this study (82.9%) and all healthy horses were natives of California.
Of all 50 diseased equids, 54% (n = 27/50), 22% (n = 11/50), and 6% (3/50) had a myopathic, neuropathic, and mixed process based on muscle histological analysis, respectively. Evidence of cardiomyopathy was seen on 8% (n = 4/50). Of 27 horses with a myopathic
Page 18 of 31
19 process, 19 had a non-inflammatory myopathy, and 8 had an inflammatory myopathy (myositis presumed associated with the presence of sarcocysts). Additionally, 3 horses had myositis and neurogenic muscle atrophy. CD8+ T-lymphocytes were the predominant cell population in skeletal muscle with evidence of myositis. Studies in mice support a critical role of CD8+ cells in the prevention of infection by S. neurona.[35] Two other studies reported myositis associated with the presence of sarcocysts.[18, 19] Horses with myositis in the present study had severe polyphasic histological lesions and marked cellular infiltration of myocytes and perimysium. These horses were lethargic, apparently painful, stiff, and reluctant to move. Two of these horses also had a swollen tongue and were dysphagic. These findings were different to those described in Quarter horses with suspected immune mediated myositis on which a cause was not identified and cellular infiltrates consisted mainly of CD4+ T-lymphocytes.[36] In humans with muscular sarcocystosis; acute and long-term persistent or intermittent myalgia, myositis, muscle wasting, arthralgia, fatigue, headache, cough from bronchospasm, rashes, facial swelling, and fever are some common manifestations.[26]
As horse meat is consumed by people worldwide, evaluating the distribution and identity of parasites in horse muscles is relevant for securing public health.[2, 4, 37] Health status of horses going to slaughter facilities is unknown, posing a risk for human disease. Humans could be either definitive or intermediate hosts to specific species of sarcocysts.[26] Sarcocystis infection has caused both, intestinal and muscular sarcocystosis in humans.[2, 26] Other forms such as cardiomyopathy and glomerulonephritis have also been reported.[26, 32] Sarcocystosis can occur by the ingestion of contaminated undercooked or raw meat, water, or fresh produce.[26] Recent outbreaks of food poisoning in humans caused by the ingestion of raw horse meat contaminated with S. fayeri have been
Page 19 of 31
20 described in Japan.[2, 4] In that study, a 15-kDa protein was extracted from horse meat containing S. fayeri.[2] This protein produced a positive enterotoxic response in rabbits characterized by diarrhea and death.[2]
Molecular analysis of the cloned parasite DNA isolated from horse muscle samples in this study were closely aligned (up to 100% identity) with sequences with GenBank samples derived from a horse meat inspection facility in Japan, where S. fayeri-caused meat poisoning has been reported.[2, 4] The high degree of polymorphisms obtained between cloned samples is likely due to variability within the high number of repetitive 18S rRNA genomic DNA copies. However, mixed infection with different S. fayeri strains is also possible. Due to the recent connection between S. fayeri-contaminated horse meat and a threat to human health, further molecular research should investigate whether toxicity from this parasite is associated with specific S. fayeri strains. Targeted meat screening for presence of toxigenic S. fayeri could provide a valuable approach for ensuring safe distribution of horse meat prior to ii reaching human consumer.
5. Conclusion Sarcocystis fayeri infection is common in young mature equids with neuromuscular disease. Sarcocystis fayeri has a moderate to high oxidative and glycolytic metabolism but has preferential establishment in low oxidative highly glycolytic myofibers of skeletal muscle. Sarcocystis fayeri infection can be associated with myopathic (non-inflammatory, inflammatory [myositis]), neuropathic, or both processes. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait
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21 deficits and in some cases dysphagia if involving the tongue and/or esophagus. Horses with myositis can be lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. Although causality between S. fayeri and neuromuscular disease has not been definitively established, the assumption of Sarcocysts spp. being an incidental finding may not be accurate for all cases. Further, as the health status of horses entering slaughter facilities may be unknown, ingestion of horse meat containing S. fayeri can pose a health hazard to human consumers.
6. References [1] Boireau P, Vallee I, Roman T, Perret C, Mingyuan L, Gamble HR, et al. Trichinella in horses: a low frequency infection with high human risk. Vet Parasitol 2000;93:309-20. [2] Kamata Y, Saito M, Irikura D, Yahata Y, Ohnishi T, Bessho T, et al. A toxin isolated from Sarcocystis fayeri in raw horsemeat may be responsible for food poisoning. J Food Prot 2014;77:814-9. [3] Jimenez-Cardoso E, Caballero-Garcia ML, Uribe-Gutierrez G, Trejo-Hernandez E, Gay-Jimenez FR. Frequency of Trichinella spiralis in blood and muscles of horses from two slaughter houses (industrial and rural) in the State of Mexico, Mexico. Vet Mex 2005;36:269-78. [4] Harada S, Furukawa M, Tokuoka E, Matsumoto K, Yahiro S, Miyasaka J, et al. Control of toxicity of Sarcocystis fayeri in horsemeat by freezing treatment and prevention of food poisoning. Shokuhin Eiseigaku Zasshi 2013;54:198-203. [5] Lele VR, Dhopavkar PV, Kher A. Sarcocysts infection in man. Indian J Pathol Microbiol 1986;29:87-90.
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22 [6] Fayer R. Sarcocystis spp. in human infections. Clin Microbiol Rev 2004;17:894-902. [7] Italiano CM, Wong KT, AbuBakar S, Lau YL, Ramli N, Syed Omar SF, et al. Sarcocystis nesbitti causes acute, relapsing febrile myositis with a high attack rate: description of a large outbreak of muscular sarcocystosis in Pangkor Island, Malaysia, 2012. PLoS Negl Trop Dix 2014;8:e2876:1-9. [8] Gunn HM, Fraher JP. Incidence of sarcocysts in skeletal muscles of horses. Vet Parasitol 1992;42:33-40. [9] Eckert J, Ossent P. Haycocknema-like nematodes in muscle fibres of a horse. Vet Parasitol 2006;139:256-61. [10] Pozio E, Celano GV, Sacchi L, Pavia C, Rossi P, Tamburrini A, et al. Distribution of Trichinella spiralis larvae in muscles from a naturally infected horse. Vet Parasitol 1998;74:19-27. [11] Pozio E, Paterlini F, Pedarra C, Sacchi L, Bugarini R, Goffredo E, et al. Predilection sites of Trichinella spiralis larvae in naturally infected horses. J Helminthol 1999;73:233-7. [12] Arriaga C, Yepez-Mulia L, Viveros N, Adame LA, Zarlenga DS, Lichtenfels JR, et al. Detection of Trichinella spiralis muscle larvae in naturally infected horses. J Parasitol 1995;81:781-3. [13] Liciardi M, Marucci G, Addis G, Ludovisi A, Gomez Morales MA, Deiana B, et al. Trichinella britovi and Trichinella spiralis mixed infection in a horse from Poland. Vet Parasitol 2009;161:345-8. [14] Mullaney T, Murphy AJ, Kiupel M, Bell JA, Rossano MG, Mansfield LS. Evidence to support horses as intermediate hosts for Sarcocystis neurona. Vet Parasitol 2005;133:27-36.
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23 [15] Cawthorn RJ, Clark M, Hudson R, Friesen D. Histological and ultrastructural appearance of severe Sarcocystis fayeri infection in a malnourished horse. J Vet Diagn Invest 1990;2:342-5. [16] Edwards GT. Prevalence of equine sarcocystis in British horses and a comparison of two detection methods. Vet Rec 1984;115:265-7. [17] Valentine BA. Pathologic findings in equine muscle (excluding polysaccharide storage): a necropsy study. J Vet Diagn Invest 2008;20:572-9. [18] Traub-Dargatz JL, Schlipf JW, Jr., Granstrom DE, Ingram JT, Shelton GD, Getzy DM, et al. Multifocal myositis associated with Sarcocystis sp in a horse. J Am Vet Med Assoc 1994;205:1574-6. [19] Herd HR, Sula MM, Starkey LA, Panciera RJ, Johnson EM, Snider TA, et al. Sarcocysts fayeri-induced granulomatous and eosinophilic myositis in 2 related horses. Vet Pathol 2015;DOI:10.1177/0300985815584073:1-4. [20] Saville WJA, Dubey JP, Oglesbee MJ, Sofaly CD, Marsh AE, Elitsur E, et al. Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses. J Parasitol 2004;90:1487-91. [21] Fukuyo M, Battsetseg G, Byambaa B. Prevalence of Sarcocystis infection in horses in Mongolia. Southeast Asian J Trop Med Public Health 2002;33:718-9. [22] Dubey JP, Lindsay DS, Fritz D, Speer CA. Structure of Sarcocystis neurona sarcocysts. J Parasitol 2001;87:1323-7. [23] Lindsay DS, Mitchell SM, Vianna MC, Dubey JP. Sarcocystis neurona (protozoa: apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development. J Parasitol 2004;90:461-5.
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24 [24] Aleman M, Watson JL, Williams DC, LeCouteur RA, Nieto JE, Shelton GD. Myopathy in horses with pituitary pars intermedia dysfunction (Cushing's disease). Neuromuscul Disord 2006;16:737-44. [25] Fischer S, Odening K. Characterization of bovine Sarcocystis species by analysis of their 18S ribosomal DNA sequences. J Parasitol 1998;84:50-4. [26] Fayer R, Esposito DH, Dubey JP. Human infections with Sarcocystis species. Clin Microbiol Rev 2015;28:295-311. [27] Hilali M, Nassar AM. Ultrastructure of Sarcocystis spp. from donkeys (Equus asinus) in Egypt. Vet Parasitol 1987;23:179-83. [28] Dubey JP, Lindsay DS, Saville WJ, Reed SM, Granstrom DE, Speer CA. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet Parasitol 2001;95:89-131. [29] Dubey JP, Streitel RH, Stromberg PC, Toussant MJ. Sarcocystis fayeri sp. n. from the horse. J Parasitol 1977;63:443-7. [30] Tinling SP, Cardinet GH, 3rd, Blythe LL, Cohen M, Vonderfecht SL. A light and electron microscopic study of sarcocysts in a horse. J Parasitol 1980;66:458-65. [31] Fayer R, Hounsel C, Giles RC. Chronic illness in a Sarcocystis-infected pony. Vet Rec 1983;113:216-7. [32] Beaver PC, Gadgil RK, Morera P. Sarcoystis in man: a review and report of five cases. Am J Trop Med Hyg 1979;28:819-44. [33] Duarte PC, Daft BM, Conrad PA, Packham AE, Gardner IA. Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis. J Vet Diagn Invest 2003;15:8-13.
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25 [34] Duarte PC, Daft BM, Conrad PA, Packham AE, Saville WJ, MacKay RJ, et al. Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses. J Parasitol 2004;90:379-86. [35] Witonsky SG, Gogal RM, Duncan RB, Norton H, Ward D, Lindsay DS. Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells. Int J Parasitol 2005;35:113-23. [36] Lewis SS, Valberg SJ, Nielsen IL. Suspected immune-mediated myositis in horses. J Vet Intern Med 2007;21:495-503. [37] Belaunzaran X, Bessa RJB, Lavin P, Mantecon AR, Kramer JKG, Aldai N. Horse-meat for human consumption-current research and future opportunities. Meat Sci 2015;108:74-81.
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26 Figures Figure 1. Sarcocyst within a myofiber of fresh frozen equine skeletal muscle on myosin ATPase reaction at various preincubation pH. (1A) Reaction at pH 9.8. Note sarcocyst within type 2 myofiber (dark color), bar 100µm. (1B) Reaction at pH 9.8. Note sarcocyst within type 1 myofiber (light, pink), bar 50 µm.
Figure 2. Succinate dehydrogenase reaction on fresh frozen equine muscle tissue. (2A) Note sarcocyst within a low oxidative myofiber (light color), bar 100 µm. (2B) Note sarcocyst within a highly oxidative myofiber (darker color) surrounded by low oxidative fibers (lighter color), bar 50 µm.
Figure 3. Disease groups based on skeletal muscle histological findings. (3A) Inflammatory myopathy. Immunohistochemistry depicting CD8+ cell infiltration, bar 100 µm. (3B) Neurogenic muscle atrophy. Note several myofibers with angular atrophy (*), bar 100 µm.
Figure 4. Formalin-fixed skeletal muscle on hematoxylin and eosin. (4A) Non-inflammatory rhabdomyolysis. Note enlarged oval myofiber containing a single sarcocyst with metrocytes (arrow head) and bradyzoites (arrow) within cyst (bar 20 µm). (4B) Myositis with combined neurogenic muscle atrophy (bar 50 µm).
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27
Table Table 1. Sarcocysts in skeletal muscle. This table depicts total muscle samples from 35 horses with neuromuscular disease (NMD, N = 61 muscles), and 36 healthy horses (Control, N = 72 muscles). Total number of myofibers containing sarcocysts is shown according to myofiber type distribution (1, 2A, 2B; high versus low oxidative) for horses with neuromuscular disease and controls. Muscle sarcocysts = number of muscles with sarcocysts, SM = semimembranosus, GLUT = gluteus medius, SCDL = sacrocaudalis dorsalis lateralis, TRI = triceps brachii, BICEPS = biceps brachii, LONG = longissimus lumbaris, EXT DL = extensor digitalis lateralis, Cr TIB = cranial tibialis, GENIO = genioglossus.
MYOFIBERS containing sarcocysts (N = 181)
NMD
MUSCLE
MUSCLE (N = 61) SM (N = 24) GLUT (N = 13) SCDL (N = 7)
Sarcocysts (N = 51/61)
1
2A
2B
High oxidative
Low oxidative
21/25
6
10
25
8
33
13/15
2
4
32
3
21
7/8
3
4
40
3
27
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28 TRI (N = 7) BICEPS (N = 2) LONG (N = 1) EXT DL (N = 1) Cr TIB (N = 1) GENIO (N = 1)
5/7
0
2
10
1
11
1/2
0
2
8
0
10
1/1
0
0
8
0
8
1/1
0
0
9
0
9
1/1
0
0
6
0
6
1/1
0
0
10
0
10
TOTAL
11 (6%)
22 (12.2%)
148 (81.7%)
27 (14.9%)
154 (85.1%)
CONTROL MUSCLE MUSCLE Sarcocysts (N = 72) (N = 1/72) SM 1/36 (N = 36) GLUT 0/36 (N = 36) TOTAL
MYOFIBERS containing sarcocysts (N = 1) 1
2A
2B
High oxydative
Low oxydative
0
0
1
0
1
0
0
0
0
0
0
0
1
0
1
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29 Table 2. Disease groups based on muscle histological findings of horses with neuromuscular disease (NMD, N = 35), and postmortem cases (PATH, N = 15). NIM = non-inflammatory myopathy, IM = inflammatory myopathy, NE = neurogenic muscle atrophy, MIX (inflammatory myopathy + neurogenic muscle atrophy), CARDIO = cardiomyopathy, NORMAL = no histological abnormalities, NA = not applicable, 1* = horse with concurrent cardiomyopathy.
DISEASED HORSES (N = 50)
NMD (N = 35)
PATH (N = 15)
TOTAL (N = 50)
NIM IM NE MIX (IM + NE) CARDIO NORMAL
18 4 10 2 NA 1
1* 4 1 1 4 5
19 (1*) 8 11 3 4 6
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30 Table 3. Percent identity between clones of Sarcocystis fayeri 18S rRNA gene products obtained from horse skeletal muscle. The heat map coloring corresponds to paired sequences that are less (light pink) to more (dark pink) similar to each other.
HORSE 1
HORSE 2
HORSE 3
HORSE 4
HORSE 5
HORSE 6
CLONE 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
1
HORSE 1 2
3
92.2 88.5 81.6 81.9 81.6 81.7 81.6 81.4 81.4 81.6 81.4 81.4 81.2 81.7 81.6 81.7 82.1
90.0 85.7 85.7 85.7 85.9 85.7 85.9 85.6 85.7 85.6 85.6 85.4 83.4 85.7 85.9 82.9
81.5 81.8 81.5 81.6 81.5 80.7 81.5 81.5 81.5 81.5 81.1 80.4 81.5 81.6 80.3
1
HORSE 2 2
3
99.2 99.7 99.8 99.7 98.8 99.5 99.7 99.5 99.5 99.3 94.8 99.7 99.8 94.4
99.2 99.3 99.2 98.3 99.0 99.2 99.0 99.0 98.8 95.1 99.2 99.3 94.7
99.8 99.7 98.8 99.5 99.7 99.5 99.5 99.3 94.8 99.7 99.8 94.4
1
HORSE 3 2
3
98.8 99.0 99.7 99.8 99.7 99.7 99.5 94.9 99.8 100.0 94.5
98.8 99.8 100.0 99.8 99.5 99.7 94.8 99.7 99.8 94.4
98.5 98.8 98.5 98.6 98.5 94.6 98.8 99.0 94.2
1
HORSE 4 2
3
1
99.8 100.0 99.3 99.5 94.6 99.5 99.7 94.2
99.8 99.5 99.7 94.8 99.7 99.8 94.4
99.3 99.5 94.6 99.5 99.7 94.2
99.1 94.6 99.5 99.7 94.4
H
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