Atypical myopathy in 2 Bactrian camels

Abstract

Atypical myopathy (AM) is an acute seasonal rhabdomyolysis seen primarily in equids, caused by the ingestion of sycamore maple samaras containing hypoglycin A (HGA) and methylenecyclopropyl-glycine (MCPG). Toxic metabolites inhibit acyl-CoA dehydrogenases and enoyl-CoA hydratases, causing selective hyaline degeneration of type I muscle fibers. Two zoo-kept Bactrian camels (Camelus bactrianus) with a fatal course of AM had sudden onset of muscle pain and weakness, recumbency, and dysphagia, accompanied by increased serum creatine kinase activity and detection in serum of HGA, MCPG, and metabolites. Medical treatment was ineffective. At postmortem examination, sycamore maple tree material was found within the first gastric compartment of the 2-y-old gelding. Although musculature was macroscopically normal, histologically, monophasic hyaline degeneration was marked within type I fibers of intercostal and hypoglossal muscles of the gelding, and in neck, tongue, and masticatory muscles of the cow. The ingestion of sycamore maple material can cause AM in Bactrian camels, and trees of the Sapindaceae family should be avoided in enclosures.

Atypical myopathy (AM), also known as sycamore poisoning and seasonal pasture myopathy, is an acute non–exercise-induced rhabdomyolysis first described in Europe in 1939.³ In Europe, AM is a seasonal disease occurring in grazing horses mainly in autumn and spring, caused by the ingestion of sycamore maple tree samaras (syn. keys or helicopter seeds) and seedlings.^15,16,20 Many Acer spp. contain hypoglycin A (HGA) and methylenecyclopropyl-glycine (MCPG), which are not toxic themselves.⁷ However, their respective activated toxic metabolites methylenecyclopropyl-acetyl-CoA (MCPA-CoA) and methylenecyclopropyl-formyl-CoA (MCPF-CoA) inhibit acyl-CoA dehydrogenases and enoyl-CoA hydratases,² which leads to impeded fatty acid β-oxidation in type I muscle fibers.¹¹ As a consequence, neutral fat accumulates in skeletal muscle fibers.^5,18,20 Fatty acids are the major energy source of slow-twitch type I muscle fibers, and hyaline degeneration with monophasic myonecrosis is selectively seen in postural, respiratory, and masticatory muscles.^5,20

In horses, the disease is characterized by sudden onset of stiffness and muscle weakness, often with concurrent pigmenturia and rapid progression to recumbency and dyspnea.^16,17 The mortality rate in horses is high with most animals dying within 72 h of the onset of clinical signs, if not euthanized beforehand.^16,17 Other clinical signs include trembling, sweating, variable degrees of muscle pain, as well as depression and signs of colic; dysphagia, cardiac arrhythmias, and colon impaction may also be seen.^16,17 Serum creatine kinase (CK) activity is usually markedly elevated.^16,19 The disease, which was formerly believed to affect mainly young grazing horses at pasture, has been described in other age ranges, other equid species, such as donkeys¹⁶ and zebras,¹ and recently also in Père David’s deer (Elaphurus davidianus),⁴ which was the first description in ruminants. Here, we describe 2 camels with a fatal course of AM, with clinical, laboratory, toxicologic, and histopathologic findings.

Two Bactrian camels (Camelus bactrianus) kept at pasture (3,500 m²) at the Zoological Garden of Neunkirchen, Germany, had clinical signs suggestive of AM 2 mo from one another. In mid-October 2016, the 2-y-old gelding had sudden onset of muscle weakness and recumbency, characterized by inability to rise. Multiple attempts indicated that neck muscle weakness failed to create the necessary momentum to rise. The heart rate was mildly elevated (72 beats/min); temperature and respiratory rate were within normal limits. Urine was macroscopically normal. The animal received intravenous fluid therapy with 0.9% isotonic saline solution (B. Braun) and electrolyte solution with 5% glucose (Sterofundin BG-5; B. Braun), as well as anti-inflammatory therapy with 15 mg/kg novamine sulfone IV (Novaminsulfon; Bela-Pharm) and 0.4 mg/kg meloxicam IM (Metacam; Boehringer Ingelheim). All dosages were based on an estimated body weight. Because of severe discomfort and lack of clinical improvement, the gelding was euthanized 6 h later. Sycamore maple trees were removed from the pasture afterward given a strong suspicion of AM.

A 5-y-old cow, kept in the same pasture, displayed drooling, dysphagia, muscle weakness, and muscle pain (clinically determined by rigidity of muscles, unwillingness to be touched in these areas, cautious movements, and grinding of teeth) 2 mo later. This was characterized by low head carriage and stumbling lasting 4 consecutive days, which then progressed to recumbency 5 h before death. Urine samples appeared macroscopically normal initially, but over the next 2 d the color gradually changed to red-brown, consistent with myoglobinuria. The cow received the same treatment as the gelding as well as supplemental feeding (soaked hay cubes, 1.5 kg supplemental feed for herbivores mixed with 10% charcoal powder per gastric tube, q8h), 2 doses of 5 mg/kg vitamin E and 0.013 mg/kg selenium IM (Vitamin E-Selen; AniMedica) on days 1 and 3, as well as 0.1 mg/kg butorphanol IV q24h (Dolorex; MSD). The cow showed mild signs of improvement (e.g. increased appetite and movement) and was hospitalized on day 3 to facilitate treatment. However, the animal died 4 d after beginning treatment.

In both camels, blood analyses revealed elevated serum activities of CK, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Additionally, the gelding had mild leukocytosis and slightly decreased vitamin E and selenium levels. The cow also had mildly increased glucose concentrations (Table 1).¹⁴ HGA, MCPG, and metabolites were analyzed by gradient chromatography–tandem mass spectrometry.^2,13 Mass spectrometric analysis showed HGA and its metabolites MCPA-carnitine and MCPA-glycine in the serum of both animals. The very high concentration of MCPF conjugates suggests that MCPG contributed significantly to the poisoning in these cases. In both camels, fatty acid β-oxidation was severely impaired as shown by high serum concentrations of butyryl-, hexanoyl-, octanoyl-, and decenoyl-carnitines, in the typical pattern of medium-chain acyl-CoA dehydrogenase deficiency. Blockade of fatty acid oxidation may be at least partially compensated by increased use of acetyl-CoA, acetoacetate, and succinyl-CoA from the breakdown of branched amino acids as an energy source. However, this reaction pathway was impeded in our cases because the degradation of valine and leucine was also severely inhibited as demonstrated by highly elevated concentrations of isobutyryl- and isovaleryl-carnitines (Table 2). Larger spectrum analysis, including long-chain acyl-carnitines, of the cow’s serum showed an accumulation of long-chain and medium-chain acyl-carnitines and complete breakdown of fatty acid β-oxidation (Table 2).

Table 1.

Laboratory results of blood parameters from camels at different times, and reference intervals.

Analyte	Male camel	Female camel			Reference interval
	Day 1 (diseased)	Day-700 (healthy)	Day 60 (diseased)	Day 61 (diseased)
CK (µkat/L)	511	1.44	54.6	68.1	0.48–8.9
(U/L)	30,600	86	3,270	4,080	729–537*
AST (µkat/L)	12.3	1.15	6.5	9.24	0.97–4.41
(U/L)	738	69	389	553	58–264*
LDH (µkat/L)	34.7	4.33	22.7	29.7	1.75–18.9
(U/L)	2,080	259	1,360	1,780	105–1,130*
Glucose (mmol/L)	13.2	6.6	8.2	17.6	4.2–15.1*
Leukocytes (×109/L)	41.2	15.6	NT	17.5	5.5–29.0*
Vitamin E (µmol/L)	1.16	NT	NT	3.02	Ø 2.62
(mg/L)					1.13†
Selenium (µmol/L)	0.98	1.84	1.23	3.00	Ø 1.27
(mg/L)					100†

Boldfaced values are outside camel reference intervals.

AST = aspartate aminotransferase; CK = creatine kinase; LDH = lactate dehydrogenase; NT = not tested, Ø = average.

^*From ZIMS (Zoological Information Management System, species360.org).

^†From Seboussi et al.¹⁴

Table 2.

Toxicologic ultra-performance liquid chromatography–tandem mass spectrometry results of blood serum (nmol/L) from camels and reference intervals from a healthy horse.

Analyte	Male camel	Female camel	Reference interval (control horse)
Hypoglycin A	503	57	Negative
MCPA-glycine	62	5	Negative
MCPF-glycine	49	225	Negative
MCPA-carnitine	36	0.1	Negative
MCPF-carnitine	2,440	647	Negative
butyryl-carnitine	7,910	2,110	<1
hexanoyl-carnitine	7,930	560	<1
octanoyl-carnitine	2,620	320	<0.1
decenoyl-carnitine	670	180	<0.1
isobutyryl-carnitine	2,330	2410	<10
isovaleryl-carnitine	5,140	1,910	<10
myristoyl-carnitine	NT	650	<10
palmitoyl-carnitine	NT	3,120	<50
stearoyl-carnitine	NT	2,020	<10
octadecenoyl-carnitine	NT	3,940	<50

Boldfaced values are outside the equine reference intervals.

MCPA = methylenecyclopropyl acetic acid; MCPF = methylenecyclopropyl formic acid; NT = not tested.

At postmortem examination, gross findings did not reveal significant pathologic changes of skeletal or cardiac muscles in either camel. In the first gastric compartment (C1) of the gelding, multiple sycamore maple samaras and leaves of different tree species were found (Fig. 1). Additionally, the animal had moderate peritoneal and pericardial effusion as well as moderate diffuse hepatic lipidosis, and moderate acute diffuse catarrhal duodeno-jejunitis with red blood-stained content. The cow also was mildly dehydrated, had multiple intestinal Trichuris sp., and mild chronic ulceration of the third gastric compartment (C3); no parts of sycamore maple trees could be identified within the ingesta, which is also frequently the case in horses with AM.⁵ Both animals were in good body condition and neither animal had diarrhea.

Figures 1–4.

Gross, histologic, and immunohistochemical findings in 2 camels with atypical myopathy. Figure 1. Sycamore maple tree samaras and leaves of various trees recovered from the first gastric compartment of the male Bactrian camel. Figure 2. Intercostal skeletal muscle of the male Bactrian camel with hyaline degeneration affecting ~80% of the myofibers. Mild mixed cellular infiltrates of macrophages and fewer neutrophils. H&E. Bar = 100 µm. Figure 3. Pterygoid muscle of the female camel with hyaline degeneration affecting ~50% of the myofibers. Moderate mixed cellular infiltrates of macrophages, fewer neutrophils, lymphocytes, and plasma cells. Scattered muscle fibers had signs of regeneration characterized by rows of large nuclei (oval). H&E. Bar = 100 µm. Figure 4. Hypoglossal muscle of male Bactrian camel with predominantly non-stained slow-twitch type 1 muscle fibers affected by hyaline degeneration (arrows), whereas SERCA1-positive type II muscle fibers were mainly unaffected. IHC for SERCA1 counterstained with Papanicolaou solution. Bar = 50 µm.

Samples from both camels were taken for histologic examination of skeletal muscles (diaphragm, semitendinosus, extensor carpi radialis, splenius, longissimus dorsi, intercostal, hypoglossal, masseter, pterygoid, and psoas major muscles), liver, kidney, spleen, esophagus, mesenteric lymph node, small and large intestine, C1, C3, lung, heart, brain, spinal cord, and, in the gelding, also brachial and sciatic nerves. The samples were fixed in 10% non-buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin (H&E). Selected sections were stained with periodic acid-Schiff (PAS) reaction using routine protocols.

The gelding had moderate-to-severe hyaline degeneration of up to 80% of muscle fibers within hypoglossal and intercostal muscles, characterized by loss of cross-striation, and swollen hypereosinophilic hyaline muscle fibers, with fragmentation and focal disruption of the endomysium (Fig. 2). Necrotic muscle fibers were surrounded by a mixed cellular infiltrate of macrophages and a few neutrophils. In the diaphragm and the skeletal muscle of the esophagus, muscle fiber degeneration was only mild. In the kidneys, globular orange-red accumulations were seen within tubular epithelial cells, interpreted as myoglobin. No further special stains were applied because of severe postmortem artifact. The small intestinal mucosa contained multiple coccidia. The liver had moderate diffuse lipid accumulation within hepatocytes. No abnormalities were observed in brachial and sciatic nerves in H&E sections.

The cow displayed moderate-to-severe hyaline degeneration of skeletal muscle fibers within the tongue, hypoglossal, splenius, and pterygoid muscles. Necrotic fibers were accompanied by moderate cellular infiltration of macrophages with fewer neutrophils, lymphocytes, and plasma cells, consistent with myophagocytosis (Fig. 3). Some regenerative muscle fibers were present, with rows of large nuclei (Fig. 3). Intercostal muscle fibers were mildly affected by hyaline degeneration. There was no evidence of glycogen accumulation (PAS-negative) or increased lipids within myofibers on H&E sections (however, lipid stains were not performed because of lack of frozen sections). Multiple nematode larvae and eggs consistent with Trichuris sp. were found within the intestinal mucosa. Liver and kidney sections were unremarkable in the cow, as were cardiac muscle fibers in both animals.

Immunohistochemistry using an antibody against SERCA1 (Clone VE121G9, dilution 1:500, Dianova; mouse mAB IgG secondary antibody, ABC detection system, Vector) to differentiate type I and II muscle fibers was performed on hypoglossal and pterygoid muscles in the gelding and cow, respectively (BenchMark XT automatic staining platform; Ventana) with 3,3′-diaminobenzidine (DAB; Millipore-Sigma) as chromogen. Mainly type I muscle fibers were affected by hyaline degeneration (Fig. 4). Immunohistochemistry using an anti-neurofilament antibody (Clone 2F11, dilution 1:400, Dako; blocking with horse serum, biotinylated horse anti-mouse IgG secondary antibody and ABC detection system, Vector) was performed on brachial nerve sections, as described previously,⁹ showed homogeneous staining of nerve fibers, indicating no significant loss of axons.

AM has not been described previously in camels, to our knowledge, and the camels described here had clinical and morphologic findings consistent with AM in equids. Both camels had a poor prognosis with no response to therapy, and a fatal course, consistent with many reports in equids.⁸ Ingestion of sycamore maple tree samaras is presumed to be the cause of the myopathy in both camels. A sycamore maple tree was present in the enclosure and parts of a sycamore maple tree were found in the C1 of the gelding. A possible higher intake of sycamore maple samaras by the camels may have contributed to development of disease because the grass was kept short by sheep newly introduced to the enclosure, making it more likely for the camels to take up the samaras while grazing. Even though the sycamore maple trees had been removed after the gelding became ill, some seeds probably remained on the large pasture, most likely resulting in continuous, but less extensive, exposure of the cow. Positive tests for HGA, MCPG, and their respective toxic metabolites in serum of both animals confirmed the diagnosis.

The affected muscle groups and type I fiber degeneration are comparable to the findings described in equids with AM.^5,6,20 In both camels, no macroscopic discolorations typical of myodegeneration were detected, even though in some muscle groups histology revealed that up to 80% of muscle fibers were affected. This is in contrast to most cases observed in equids^16,17 and deer,⁴ and should be considered during postmortem examination of suspected cases; sampling of various muscle groups independent of macroscopic changes is necessary. The myocardium, which is often affected by hyaline degeneration in horses with AM,^5,19 did not have any histologic alterations evident in the camels. Only one of the camels displayed macroscopically visible pigmenturia, therefore, this clinical sign may not be as consistent as in horses with AM.^16,17 Lack of pigmenturia may be the result of the difference in disease course of both camels, given that urine discoloration was only observed several days after onset of clinical signs in the cow. However, the male camel did have orange-red accumulations interpreted as myoglobin within tubular epithelial cells of the kidney, indicating that pigmenturia would have appeared soon had the animal not been euthanized. The female camel also had mild hyperglycemia, which is commonly described in horses.¹⁷ This is in contrast to human Jamaican vomiting sickness, caused by the ingestion of HGA-containing ackee fruits, and laboratory rats administered HGA or MCPG, in which hypoglycemia is described.^10,12

The gelding had mildly decreased vitamin E and selenium concentrations. These deficiencies cannot be totally excluded as contributing factors but are very unlikely to be the underlying cause of the myopathy, given that vitamin E and selenium deficiency as a cause of nutritional myopathy is characterized by multifocal polyphasic myonecrosis, whereas AM is associated with multifocal monophasic myonecrosis, as seen in our cases.⁶ Further studies should focus on the overall susceptibility of non-equid species to AM. Despite sycamore maple trees generally being considered a safe plant species, animal holding facilities should, as a preventive measure, assess the flora in animal enclosures and consider the removal of Acer spp. containing the toxins.⁷ Both Acer platanoides (Norway maple) and Acer campestre (field maple), which are also commonly found in Europe, have tested negative for toxic compounds causing AM.^7,18