Neurologic disease produced by Rathayibacter toxicus-derived corynetoxins

Corynetoxins, members of the tunicamycin group of antibiotics, are produced by the bacterium, Rathayibacter toxicus. They cause a severe neurologic disorder in domestic livestock, are hepatotoxins, and can damage retinal photoreceptors. For these toxins to be ingested by livestock, the bacterium must first be transported onto host plants by adhering to nematode larvae. In the infected seed heads, bacterial galls (gumma) then form. While corynetoxicity occurs most commonly in Australia, it has occurred sporadically in other countries and, due to the widespread global distribution of the bacterium, nematode, and host plants, there is great potential for further spread, particularly as the range of host plant species and nematode vectors identified for R. toxicus is increasing. Since many animal species are susceptible to corynetoxins poisoning, it is likely that humans would also be vulnerable if exposed to these potent, lethal toxins.


Introduction
Corynetoxin poisoning is a severe, and frequently fatal, neurologic disorder of domestic livestock, principally sheep and cattle but, occasionally, also horses. 1 These toxins are also true or intrinsic hepatotoxins, 1 and cause retinal degeneration in a range of animal species. 2,3This disease is caused by a nematode (Anguina sp.)-bacterium (Rathayibacter toxicus) complex, which parasitizes host plant species consumed by livestock.8][9] It has the potential to spread much more widely, especially in the United States, due to the broad distribution of R. toxicus, vector nematodes, and identified (and potential) host plant species. 10There is also a real threat to food safety and human health from corynetoxin contamination of the food supply, either via grain or consumption of products from subclinically affected livestock, 10 particularly as these potent toxins have been shown to cause neurologic disease, and often death, in a wide range of animal species. 1

Geographic distribution of corynetoxicity
At present, the naturally-occurring disease produced by corynetoxins occurs principally in Australia and, to a much lesser extent, South Africa, and the potential to spread further in these two countries, and more widely, is considerable. 1In the United States, for example, R. toxicus is listed as a Plant Pathogen Select Agent, mainly due to its potential to damage forage-consuming domestic livestock.The threat of introduction, and establishment, of R. toxicus in the United States is very high due to the presence of susceptible grasses and potential nematode vectors.R. toxicus may also colonize, and produce corynetoxins, in a wide range of cereals consumed by humans.This bacterium is widely distributed, the host plants are common worldwide, and R. toxicus is capable of being transported onto plants by nematodes other than those currently associated with disease. 10,11n Australia, the first suspicion of a nematode-bacterium complex causing sheep mortalities occurred in 1956 in the mid-north of the state of South Australia, this disease interaction being confirmed in a 1967 outbreak. 12The disease spread slowly at first, then more rapidly, especially when it appeared in the south-west of Western Australia in 1971. 13ost Australian outbreaks occur during summer grazing of the host pasture, annual ryegrass (Lolium rigidum), which is a major component of improved pastures in these States.This pasture association has led to the common designation of the disease as Annual Ryegrass Toxicity or ARGT. 1 In 1991, in Australia, R. toxicus was also found to be responsible for a severe neurologic disorder with high mortality in sheep and cattle grazing annual beard grass (Polypogon monspeliensis) in flood-prone areas of the south-east of South Australia, 14,15 and blown grass (Agrostis avenacea) on the floodplains of northern New South Wales. 15,16Annual ryegrass is not a prominent pasture species in either of these regions.Coryetoxicosis has now been found in over 10 million hectares of farmland in Australia. 11In 1985, toxic tunicaminyluracil antibiotics also caused deaths of Australian pigs fed water-damaged cereal grain. 17,18orynetoxicity has been described in sheep and cattle, 7 and horses, 19 in South Africa, the causative R. toxicus probably being introduced from Australia in contaminated ryegrass. 7oreover, an outbreak occurred in Japan in sheep and cattle fed contaminated oat hay imported from Australia. 20 similar neurologic disorder associated with Anguina sp.nematodes and a Rathayibacter-like bacterium has been sporadically reported in cattle and sheep grazing Chewing's fescue (Festuca nigrescens) in Oregon, USA in 1945, 1949 and 1961.8,9 The intermittent nature of these Oregon outbreaks has been attributed to the annual burning of fescue pastures, which eliminates the causative nematode populations.9 Less well documented cases of corynetoxicosis have also been reported from several other countries.11 It is possible that climate change could influence the geographic distribution of this toxic disease by, for example, positively or negatively affecting the growing conditions of the host plants, life cycle of the vector nematodes, or proliferation of the causative bacterium.However, to date, any potential effects are speculative, since there is no published material that has addressed this issue.
The nematode-bacterium complex required for corynetoxin poisoning R. toxicus, previously termed Clavibacter toxicus and Corynebacterium rathayi, is a toxin-producing, Grampositive, nematode-vectored, phytopathogenic bacterium.The Rathayibacter genus consists of at least 9 species that require plant parasitic nematodes of the Anguinidae taxon to be vectored to plants. 11,21The complex lifecycle of R. toxicus, leading to production of corynetoxins, involves this bacterium, a nematode, the host plant and, probably, an associated bacteriophage.Two other Rathayibacter species, R. iranicus and R. sp.EV are suspected, but not yet proven, to produce corynetoxins. 10lant species that become toxic are colonized soon after germination by Anguina sp.nematode larvae, which modify the floret primordia of the plant to produce hollow, flaskshaped galls (Figure 1).The galls are an enlarged, reactive mass induced by larval invasion, and replacement of, the intact seed.Nematodes of the genus Anguina are obligate plant parasites of global distribution. 1While Anguina funesta prefers annual ryegrass (Lolium rigidum), a pasture plant introduced to Australia in 1880, it can also parasitize other grasses, such as annual beard grass (Polypogon monspeliensis) and blown grass (Agrostis avenacea). 14,16nguina sp.nematodes are an essential vector for R. toxicus, which cannot penetrate pasture grasses unaided.While A. funesta is the preferred vector of R. toxicus, other Anguina sp.nematodes (such as A. paludicola) can also serve as vectors.Moreover, R. toxicus may potentially colonize, and produce corynetoxins in, a wide range of cereals consumed by humans, but they are usually incidental hosts near heavily infected annual ryegrass pastures.After attaching to the surface of infective, second-stage nematode larvae in the soil, R. toxicus damages, then inserts itself beneath, the nematode cuticle.Bacteria are thus carried into the nematode galls, where they multiply.If conditions favor bacterial proliferation, the nematodes atrophy and die, and a bacterial gall is formed.Only bacterial galls are toxic.Bacterial galls are bright yellow (Figure 2) and termed gumma, for they are composed of a gummy or sticky exudate. 10,22here is a considerable delay between the time galls are colonized by bacteria and when they become toxic to livestock.It is estimated to take 10-15 years after the introduction of the infection into a region before toxin quantities lethal to livestock are produced. 1Most infected plants do not show any abnormality, thus the absence of a visible yellow slime (gummosis) does not mean that a plant is free of R. toxicus.Corynetoxins are not found in all bacterial galls, and are usually produced as infected plants become senescent and bacteria reach their peak biomass. 10nvolvement of a bacterial virus (bacteriophage) is also considered to be important for toxin production. 23In Australia, where corynetoxicosis is most prevalent, the disease occurs during summer grazing, but it can occur at any time of the year if livestock are fed toxic hay.Livestock deaths occur throughout the summer period until the opening autumn rains arrive. 1 Corynetoxins R. toxicus produces a mixture of 16 closely related, and structurally similar, toxins, which are designated corynetoxins.The addition of a bacteriophage (NCPPB3778) to R. toxicus cultures correlates with the production of corynetoxins, but it is uncertain whether this bacteriophage is always required for synthesis of corynetoxins in natura. 10,23][6] Tunicamycin, which is closely structurally related to, and biologically indistinguishable from, corynetoxins was originally isolated from the actinomycte, Streptomyces lysosuperificus. 24The neurologic disorder in livestock is the only naturally-occurring disease caused by tunicaminyluracil toxins. 1 Corynetoxins are heat stable and highly toxic.Glycolipids are poor immunogens and natural immunity against corynetoxins has never been reported in affected animal species.5][6] These toxins are also lethal for all species tested to date, including sheep, cattle, horses, pigs, chickens, guinea pigs, rats, and mice.In all species, except mice, death is preceded by neurologic disturbance. 1  Both corynetoxins and tunicamycin share a common core containing the nucleoside, uracil, to which is attached the unique 11 carbon dialdose aminosugar, tunicamine.Tunicamine, in turn, is attached to an N-acetyl-glucosamine.The different corynetoxins and tunicamycin differ from each other only in the nature of the fatty acid chain linked to the amino group at C-10 0 of tunicamine. 5orynetoxins cause specific and potent inhibition of lipid-linked N-glycosylation of glycoproteins, including enzymes, hormones, cell membranes, extracellular matrix components, and membrane receptors. 4Their toxicity in animals is probably the result, either directly or indirectly, of the depletion or impaired synthesis of essential N-glycosylated glycoproteins. 1 Since glycoproteins are widely distributed in tissues, and have myriad functions, their inhibition by corynetoxins impedes numerous cellular functions and the potential effects of their toxicity in animals are diverse. 25any proteins are glycosylated, containing covalently linked carbohydrate chains as part of their molecular structure.The carbohydrate may have a non-specific function in the stabilization of protein conformation or protection from proteolysis.In the synthesis of N-glycosaylated proteins, the oligosaccharide is first assembled on a lipid carrier and, in the presence of sufficient amounts of corynetoxin, this lipid-linked oligosaccharide cannot be formed, and the protein is not glycosylated.N-linked glycosylation occurs only in the rough endoplasmic reticulum (RER), and nearly all proteins that pass through the RER become glycosylated in some form.In the absence of the carbohydrate moiety, the protein conformation may be altered, leading to decreased solubility, increased vulnerability to denaturation and proteolysis, and an inability to be secreted.In some glycoproteins, the carbohydrate component is essential for normal function.In the central nervous system, N-glycosylated glycoproteins form 85-90% of protein-bound carbohydrate, and glycoproteins are present in substantial amounts in synaptic membranes and vesicles, transmitter receptors, myelin, neurons, and glia.However, the precise biological roles of many of these glycoproteins in the brain are still incompletely understood. 26,27turally-occurring and experimental corynetoxicity

Clinical signs
Livestock losses tend to be highest in sheep, although economic loss from cattle deaths can be substantial.After the introduction of sheep to toxic pastures, neurologic signs may be manifest within 1-2 weeks, but can take up to 12 weeks.All ages are affected, and morbidity and mortality rates are frequently high. 1 When an affected flock is driven, some sheep collapse in sternal or lateral recumbency.They often show muscle tremors, opisthotonus (Figure 3), head nodding, tetanic and clonic convulsions (Figures 3 and 4), limb extension, grinding of the teeth (bruxism), nystagmus, and ptyalism.Sheep may regain their feet after several minutes, and stagger away with a stiff-legged, jumping, or swaying gait.These sheep often wander aimlessly, exhibiting depression and ataxia.Abortion can occur in up to 10% of surviving pregnant ewes.Removal of stock from toxic pastures usually results in cessation of neurologic

Pathology and pathogenesis
At autopsy, in sheep dying from corynetoxicity, the liver is yellowish and friable, and the carcase is sometimes mildly icteric.Hemorrhages are frequently found in the gallbladder and, less commonly, in the rumen, small intestine, kidney, and cervical musculature.Petechial epicardial, and ecchymotic endocardial, hemorrhages are regularly found and there is sometimes clear fluid in serous cavities.Meninges are congested. 1hile corynetoxicity presents as a severe neurologic disorder, histomorphologic changes in the brain are mild, inconsistent, and often absent; none are pathognomonic.Lesions are most commonly found in the cerebellum.The most consistent microscopic finding is perivascular deposition of eosinophilic, proteinaceous fluid in the cerebellar meninges.9][30][31] In terminally convulsive sheep, focal spongy degeneration may be found in the white matter of the dorsolateral thalamus, 32 but this lesion can also occur in other ovine convulsive disorders. 32ltrastructural studies have been useful in elucidating the pathogenesis of brain lesions occurring in corynetoxicity, particularly in tunicamycin-treated guinea pigs.In this species, brain (and liver) lesions closely resemble those found in sheep, but are more severe and widely distributed, particularly in the brain.Brain damage in guinea pigs also has a predilection for the Purkinje cell and granular layers of the cerebellum, and degenerative parenchymal lesions, especially focal granular layer necrosis, are largely referrable to deleterious changes in microvessels. 28,29Several mechanisms of microvascular occlusion (Figure 5), which result in impaired perfusion and focal parenchymal ischemic-hypoxic injury (Figure 5), are found: (1) severe endothelial damage, with desquamation of these effete cells into the capillary lumen; (2) marked dilatation of cisternae of endothelial RER, which often causes partial luminal stenosis, and (3) severe swelling of astrocytic end-feet as a result of increased vascular permeability, which appears to collapse the enclosed capillary. 29In guinea pigs given tunicamycin, emboli derived from damaged hepatocytes also contributed to capillary obstruction in the brain. 33orynetoxins conform to the classification of intrinsic (predictable or true), as opposed to idiosyncratic, hepatotoxins, since the liver damage they produce is dosedependent, predictable, and reproducible in experimental animals, and the underlying injury mechanisms are typically at last partially understood. 34Microscopic hepatic changes in sheep or guinea pigs exposed to corynetoxins or tunicamycin are characterized by diffuse vacuolation of hepatocytes, due to marked dilatation of RER and lipid accumulation; scattered foci of individual hepatocellular necrosis; formation of hepatocyte-derived apoptotic bodies; and bile ductule hyperplasia. 33,35While these hepatic changes are etiologically non-specific, the finding of unusual, concentrically laminated, and probably proteinaceous, concretions at the fine structural level in dilated hepatocyte RER is more specific for corynetoxin poisoning.The pattern of hepatocellular damage in sheep and guinea pigs given corynetoxins/tunicamycin is diffuse. 33,35However, for reasons yet to be determined, liver injury in guinea pigs becomes predominantly periportally distributed by 72 h post-injection of tunicamycin, 33 a zonal pattern usually caused by direct toxins. 34Death of hepatocytes is a mix of apoptosis and necrosis 37 and is manifested by elevated plasma levels of liver-specific enzymes and ammonia, the latter not considered to be sufficiently increased to cause hepatic encephalopathy.Loss of ribosomes on the damaged RER results in decreased serum protein levels. 33,35he pathophysiological mechanisms by which drugs injure the liver generally involve their interaction with, or disruption of, cellular targets 34 which, in the case of corynetoxins, is the RER.Marked distension of the cisternae of RER is due to the inhibitory action of corynetoxins/ tunicamycin on protein glycosylation, a process which occurs in this organelle. 26,27The other cause of hepatocyte vacuolar degeneration is accumulation of lipid, some of this fatty change being attributed to inanition, which develops soon after toxin exposure. 33,35However, since cultured hepatocytes exposed to tunicamycin also accumulate lipid, 38 this agent may directly damage hepatocytes.Moreover, the ER stress caused by tunicamycin disrupts lipid regulation, leading to fatty change, the RER being the major site of lipid metabolism in hepatocytes. 39Fatty liver disease is also abetted by oxidative stress, tunicamycin increasing the formation of reactive oxygen species and suppressing endogenous antioxidant activity. 39unicamycin has been shown to cause severe retinal photoreceptor degeneration in guinea pigs, cats, rats and mice. 2,3Since brain lesions are often minimal, and liver pathology etiologically non-specific, in corynetoxinpoisoned ruminants, the occurrence of similar ophthalmic injury in these livestock species could be diagnostically useful. 3

Diagnosis and treatment
Gross pathologic changes in the brain of sheep dying from corynetoxicity are scant, and liver lesions, although often severe and consistently present, are, nevertheless, not pathognomonic for corynetoxicosis.9][30] Ultrastructural examination of the brain reveals several mechanisms of microvascular obstruction and compromised perfusion, leading to localized ischemic-hypoxic parenchymal injury. 29In the liver, there is some specificity in the markedly dilated hepatocyte RER 33,35 and, more particularly, the unusual concretions in this distended organelle. 35e most common ancillary laboratory examinations used for corynetoxicity diagnosis are examination of pasture seed heads for nematode and bacterial galls and a bacterial enzyme-linked immunosorbent assay (ELISA) for the presence of R. toxicus.There is also an ELISA for corynetoxins, but it requires an arduous extraction process and is more expensive and less sensitive than a bacterial ELISA. 40,41A highly antigenic protein secreted by R. toxicus, but not other Rathayibacter spp., has been identified in vitro.However, whether this high level of expression occurs under natural field conditions is unknown. 42reatment for corynetoxin-poisoned ruminant livestock is very limited and often impractical, although less severely affected animals may recover with supportive treatment.The most prudent management strategy at present is to move stock as expeditiously as possible to clean, non-toxic pasture, and the most common preventive measure is examination of suspected toxic pastures for nematode and bacterial galls before allowing stock access. 1,40,41

Declaration of conflicting interests
The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figure 3 .
Figure 3.A sheep dying from corynetoxicity in South Australia has excavated the soil with paddling convulsions.
signs and mortality after about a week.Clinical signs in cattle resemble those in sheep.1