Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein and formation inclusions in neurons or oligodendrocytes (1). A recent study by Tayebi and colleagues demonstrated that Phalaris toxicity (PT) led to an accumulation of neuromelanin and aggregated α-synuclein in the central and peripheral nervous system of kangaroos after intoxication with tryptamine-alkaloid-rich Phalaris spp. pasture plants (2). PT is known to affect sheep as well as cattle and horses at pasture; this widely used and studied perennial pasture plant can become toxic after heavy rainfall, which follows a prolonged period of dry weather (3). Sheep with Phalaris staggers, a neurologically progressive and fatal form of PT (4–8), exhibit gait abnormality, hyperexcitability, muscle tremors, incoordination, limb paresis, convulsions, recumbency, collapse/falling, and death (6, 9). It was previously reported that sheep affected with Phalaris staggers show neuropathological lesions that included intense macroscopic green discoloration of the gray matter of several brain regions, including thalamus, brainstem, and ventral horns of the spinal cord and microscopic intraneuronal brown pigmentation (3).

It has long been believed that the serotonin-analogous tryptamine alkaloids (TA) cause Phalaris staggers in sheep (7, 9, 10). TA causes Phalaris staggers due to competitive binding to the serotonergic receptors (10). A historically important landmark study reported by Gallagher et al. (11) demonstrated that sheep developed clinical signs of Phalaris staggers after administration of TA (11), specifically N,N-dimethylated tryptamine alkaloids of Phalaris tuberosa, which induced violent convulsions with spasticity. Similarly, the synthetic MPTP drug, structurally analogous to TA-derivatives (12), caused parkinsonism in humans and monkeys (13–16), an indication that alkaloids might be directly or indirectly implicated in parkinsonism syndromes (17, 18).

In this study, we demonstrate the accumulation of aggregated α-synuclein and neuromelanin in the central nervous system (CNS) of sheep naturally affected by Phalaris staggers. This is the first study to link Phalaris plant poisoning in sheep with α-synucleinopathy classically associated with parkinsonism and some other human neurodegenerative disorders.

Phalaris plants are widely cultivated as pasture grasses because they are robust and grow well on varying soil types. The syndrome of Phalaris staggers is recognized worldwide, and farming communities manage their pastures to minimize losses, but at times cases occur. For the purposes of this investigation, from the Elizabeth Macarthur Agricultural Institute (EMAI), New South Wales (NSW) Department of Primary Industries, we collected archived tissues from six mixed-breed juvenile to 4.5-year-old sheep. Dead or moribund sheep had been submitted for diagnostic postmortem examination between January and November 2017. They were all known to graze on farms across NSW (Table 1) with a history of PT and were exposed to Phalaris spp. After access to Phalaris pastures for some period, all six sheep presented to the referring large animal veterinarian with a variety of neurological signs, which included ataxia, paralysis/paresis, falling, tremor, circling, recumbency, and eventually death (Table 1). After gross postmortem examinations and histopathological assessment at the EMAI, all were diagnosed with chronic Phalaris neurotoxicity based on plant exposure, clinical presentation, and neuropathological changes (Table 1). In all cases, extensive diagnostic investigations ruled out other neurological and non-neurological disorders. The five non-Phalaris-exposed control sheep were cases submitted to EMAI between March and September 2018. Most were classified as adults and some Merino; 2/5 were ewes with pregnancy toxemia, whereas other miscellaneous diagnoses were made. One sudden death case remained undiagnosed, but Phalaris neurotoxicity was ruled out.

Phytochemicals are structurally diverse biologically active compounds of plant origin that possess various beneficial and toxic properties toward synucleinopathies (21). Here, we describe the effect of alkaloids from Phalaris on the development of pathology in sheep. We have recently reported cases of synucleino-neuromelanopathy in eastern gray kangaroos affected with PT after ingestion of tryptamine-alkaloid-rich Phalaris spp. In this study, we also describe cases of α-synucleino-neuromelanopathy in sheep associated with PT after grazing on Phalaris pastures. These animals displayed typical neurological deficits previously described for sheep affected with Phalaris staggers (3, 4, 8, 9, 22) and included ataxia, paralysis/paresis, falling, tremor, circling, recumbency, and death. These neurological deficits implicate the caudal brain stem and spinal cord (ataxia, paresis/paralysis, and potentially circling) and possibly further areas of the CNS. Microscopic assessment of H&E-stained brain sections from the sheep confirmed the neuronal accumulation of the neuromelanin brown pigments in these affected animals in the cerebral cortex, thalamus, brainstem, and spinal cord, whereas unaffected animals remained neuromelanin-free. The clinical findings mirror those described previously by other authors for sheep (8, 9, 23–28) and the more recently described lesions in kangaroos (2), which showed conspicuous accumulation of neuromelanin pigments in the neurons of the central and enteric nervous systems, implicating a causative role for the Phalaris plants whose neurotoxicity is well known (6, 8). Moreover, and comparable to our findings in kangaroos (2), the Warthin–Starry reaction is consistent with these neuronal pigments being neuromelanin. In humans, the presence of aggregated neurotoxic species of α-synuclein in the CNS signifies a hallmark of Parkinson's disease (PD) and other related neurodegenerative disorders (29–33). The α-synuclein appearance was previously described as ovoid and fusiform to thread-like aggregates in human PD (29–33). A similar appearance was described in kangaroos affected with PT (2). Although the α-synuclein presentation was morphologically similar in some neurons of sheep affected with PT, the majority presented as fine punctate and conspicuous diffuse perikaryal pattern. This differential expression might suggest an acute clinical phase of the disorder, but whether this is the case needs further confirmation. To rule out whether this difference in the α-synuclein presentation was artifactual and might have been due to α-antibody specificity, staining with the 97/8 rabbit anti-human α-synuclein antibody (19) was confirmed with the widely used and validated MJFR1 rabbit anti-human α-synuclein antibody raised against human recombinant full-length α-synuclein and mapped to amino acids 118–123 (VDPDNE). Of note, a high degree of sequence homology was found between α-synuclein of sheep and α-synuclein of humans (~96%) (34).

The presence of aggregated α-synuclein in neurons of sheep brains was demonstrated in several areas, including the cerebral cortex, thalamus, brainstem, and spinal cord, and near neuromelanin pigments. Both peracute (cardiac) and delayed, chronic (CNS) toxicities are associated with Phalaris consumption. Bourke (3) showed that one neurotoxic alkaloid does not cause cardiac syndrome. The extensive distribution of α-synuclein in different brain regions suggests a potential effect on multiple neuronal populations. It is common for neuropathological studies to reveal more widespread neural injury than indicated by the clinical signs. Gradual poisoning by ingestion might involve progressively wider CNS accumulation, which in time and with longer survival, might mirror neuropathological changes associated with PD (35). However, in a study of α-synuclein accumulation in scrapie-affected sheep and goats, Adjou et al. (36) found widespread granular deposits in the brains, especially the hippocampus and cerebellum. Astrocytosis noted in areas bearing only α-synuclein deposits was believed to suggest a role for synucleinopathy in sheep and goat scrapie. Of interest, in this second neurological disorder of sheep (and goats), α-synuclein deposits in the brain were global, and furthermore, Lewy body formation was not described. All sheep included in these studies and affected with Phalaris staggers showed microglial activation and gliosis.

It was previously reported that the molecular mechanisms underlying the degeneration of axons are distinct from those affecting neuronal bodies. It was confirmed that axonal loss precedes neuronal cell death in PD and other neurodegenerative disorders (37). Moreover, and despite the lack of conclusive evidence related to the molecular mechanisms associated with α-synuclein aggregates accumulation in human idiopathic PD and other parkinsonism syndromes, tryptamine-alkaloids analogous MTPP was shown to cause nigrostriatal toxicity and leads to chronic parkinsonism in humans and monkeys (13–16).

In this report, we described cases of synucleinopathies in sheep, which appear to be as a consequence of ingesting alkaloid-rich Phalaris spp. This study helped characterize the sheep as a higher mammalian model for examining the consequences of progressive CNS synucleinopathy with relevance to parkinsonism. Akin to current methodology to evaluate brain β-amyloid plaques with (18)F-labeled tracers for positron emission tomography, it may be possible in the sheep brain to image α-synuclein accumulation. Most importantly for the veterinary profession, this provides an additional new postmortem diagnostic tool for ovine Phalaris staggers.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Ethical review and approval was not required for the animal study because Sheep were submitted for routine diagnostic post-mortem examination to the Department of Primary Industries and as such are not subject to animal ethics guidelines.

MT: conceived and designed experiments and wrote the manuscript. PP, UH, and RK: performed experiments. MD: designed experiments and revised manuscript. BS: conceived experiments and revised manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the University of Western Sydney Start Up funds to MT.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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