Flow-mediated K+ secretion in horses intoxicated with lolitrem B (perennial ryegrass staggers)

AIM: To investigate the effects of lolitrem B intoxication on renal K⁺ secretion in response to increased tubular flow rates.

METHODS: Results are derived from a repeated measure pilot study of seven horses fed non-perennial ryegrass feed for a week prior to exposing them to perennial ryegrass seed and hay that contained an average of 2 ppm lolitrem B. At the end of the control and treatment period frusemide (1 mg/kg I/V) was administered and serial fractional excretion of K⁺(FEK⁺) and fractional excretion of Na⁺(FENa⁺) calculated. Baseline concentration of aldosterone in plasma, serum K⁺concentration and feed K⁺ concentration were also compared.

RESULTS: Key findings included a reduced change in FEK⁺ from 0 to 15 minutes in response to frusemide administration (p=0.022, Wilcoxon signed-rank test) and a reduced baseline concentration of aldosterone in plasma (p=0.022, Wilcoxon signed-rank test) during the treatment period compared with the control.

CONCLUSIONS: Results suggest that lolitrem B intoxication reduced flow-mediated K⁺ secretion and interfered with aldosterone production or secretion. However, further investigation is required to validate these findings and to further elucidate the underlying pathophysiology.

CLINICAL RELEVANCE: Lolitrem B intoxication in horses may cause disruption to electrolyte handling in addition to neurological deficits.     

Lolitrem B residue in fat tissues of cattle consuming endophyte-infected perennial ryegrass straw.

Lolitrems are neurotoxins found in endophyte-infected perennial ryegrass. Lolitrems, primarily lolitrem B, are the causative agents of ryegrass staggers in livestock. To guarantee the safety of meat produced from cattle consuming endophyte-infected perennial ryegrass, lolitrem B concentrations in tissues of Japanese Black cattle were determined using high-performance liquid chromatography. Lolitrem B was not detected in muscle, liver, kidney, or cerebrum of a Japanese Black cow with signs of ryegrass staggers. In contrast, perirenal fat contained 210 ppb lolitrem B. Three cows that received half as much perennial ryegrass straw as the cow with ryegrass staggers showed no clinical signs of ryegrass staggers. However, low concentrations of lolitrem B (less than 150 ppb) were detected in their fat tissue. These observations indicate that human exposure to the neurotoxic effect of lolitrem B through beef is unlikely. The amount of lolitrem B consumed by cattle can be estimated by the determination of lolitrem B in fat tissue.     

Correlation of ergovaline and lolitrem B levels in endophyte-infected perennial ryegrass (Lolium perenne).

The varieties of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Neotiphodium lolii contain several classes of toxic alkaloids, including ergopeptide alkaloids and lolitrem alkaloids. Lolitrem B, a potent tremorgen, is generally considered to be the predominant alkaloid in endophyte-infected perennial ryegrass. Ergovaline, a vasoconstrictor normally associated with endophyte-infected tall fescue (Festuca arudinacea), is also present in endophyte infected perennial ryegrass. Clinical signs of animals ingesting endophyte-infected perennial ryegrass are consistent with the presence of lolitrem B. However, clinical signs normally associated with ergovaline poisoning are not usually observed in animals ingesting endophyte-infected perennial ryegrass. A survey was conducted to quantitate both lolitrem B and ergovaline in 459 perennial ryegrass straw samples received at the Oregon State University College of Veterinary Medicine. Samples were analyzed for each alkaloid using separate high-performance liquid chromatography analyses. A strong positive correlation between the 2 alkaloids (r2 = 0.7335) was observed, especially in the samples containing <3,000 ppb (ng/g) lolitrem B. The threshold levels above which clinical signs typically occur are 2,000 ppb lolitrem B and 300-400 ppb ergovaline. All of the samples analyzed contained <425 ppb ergovaline.     

The effect of glyphosate, paraquat and paclobutrazol on lolitrem B levels in endophyte-infected perennial ryegrass

Two herbicides (glyphosate and paraquat) and a plant growth regulator (paclobutrazol) were applied to endophyteinfected (Acremonium lolii) perennial ryegrass swards. Subsamples of these swards were then chemically analysed at intervals up to 28 days later for lolitrem B, the compound responsible for perennial ryegrass staggers in domestic livestock. Glyphosate and paclobutrazol had no effect on lolitrem B concentrations. Paraquat applications decreased lolitrem B concentrations in the herbage. Because none of the chemicals tested increased the concentration of lolitrem B in the herbage, they are unlikely to be directly implicated in perennial ryegrass staggers in grazing animals.     

Evaluation of perennial ryegrass straw as a forage source for ruminants

Two experiments were conducted to evaluate perennial ryegrass straw as a forage source for ruminants. Experiment 1 evaluated digestion and physiological variables in steers offered perennial ryegrass straw containing increasing levels of ergot alkaloid, lolitrem B. Sixteen ruminally cannulated Angus x Hereford steers (231+/-2 kg BW) were blocked by weight and assigned randomly to one of four treatments. Steers were provided perennial ryegrass straw at 120% of the previous 5-d average intake. Before straw feeding, soybean meal was provided (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Low (L) and high (H) lolitrem B straws (<100 and 1,550 ppb, respectively; DM basis) were used to formulate treatment diets: 100% L; 67% L:33% H; 33% L:67% H; 100% H (DM basis). Intake and digestibility of DM and OM, and ruminal pH, total VFA, and NH3-N were not affected by increasing lolitrem B concentration. Ruminal indigestible ADF (IADF) fill increased linearly (P = 0.01) and IADF passage rate decreased linearly (P = 0.04) as lolitrem B increased. Experiment 2 evaluated performance and production by 72 Angus x Hereford cows (539+/-5 kg BW) consuming perennial ryegrass straw containing increasing lolitrem B during the last third of gestation. Cows were blocked by body condition score and randomly assigned to one of three treatments. Cows were provided perennial ryegrass straw ad libitum and supplemented with soybean meal (0.1% BW; CP basis) to meet the estimated requirement for degradable intake protein. Mixtures of a L and H lolitrem B straw (467 and 2,017 ppb, respectively) were used to formulate treatment diets: 100% L, 50% L:50% H, 100% H (DM basis). Thirteen of 24 cows on the 100% H treatment exhibited signs of ryegrass staggers and were removed from the study; nevertheless, lolitrem B concentration did not influence pre- or postcalving weight or body condition score change. These data suggest that feeding perennial ryegrass straw containing up to 1,550 ppb lolitrem B (DM basis) did not adversely affect nutrient digestion or physiological response variables in steers. However, providing straw with a lolitrem B concentration of approximately 2,000 ppb (DM basis) resulted in 54% of cows exhibiting signs of ryegrass staggers. These data suggest that blending straws with a high (>2,000 ppb) and low (<500 ppb) concentration of lolitrem B can be a successful management practice.     

Rhinopneumonia or mycotoxin intoxication? Neurologic phenomena in horses from a riding school

In the course of several days most of the 40 riding-school horses turned out in paddocks developed ataxia of variable severity. Five of these horses showed severe ataxia and tremors, became paralyzed and were euthanized. Eleven privately-owned horses which were stabled on the same premises showed no clinical signs. The most likely diagnosis seemed to be the 'neurological form of EHV1', although the signs were not entirely typical. A few weeks later a second outbreak occurred among the riding-school horses and one of the privately-owned horses also showed signs of ataxia. In the meantime it had been shown that EHV1 titers in paired serum samples had not increased and that the cerebrospinal fluid of one of the severely affected horses was normal. Toxicological examination of hay, delivered just before the first outbreak and stored for the winter, showed a significantly increased concentration of lolitrem B mycotoxin (5-6 mg/kg). The hay appeared to have been made of ryegrass used for lawns and playing fields. Retrospectively it became probable that this hay occasionally been fed to the horses just before the onset of clinical problems. It is concluded that the horses showed the 'ryegrass-stagger syndrome'.     

Detection of endophyte toxins in the imported perennial ryegrass straw

From 1997 to 1999, 29 cases of disorders were detected in cattle and horses that had been fed ryegrass straw imported from the U.S.A. These animals showed symptoms resembling ryegrass staggers and the clinical signs disappeared after removal of the straw. Endophytic hyphae were detected in the seeds of all straw samples that were responsible for the clinical cases. Lolitrem B concentrations in the straw ranged between 972 and 3740 ppb. Ergovaline concentrations were between 355 and 1300 ppb. Even though the concentrations of lolitrem B were lower than the toxic threshold proposed by Oregon State University in better part of the cases, our observations suggest the possibility that lolitrem B lower than the proposed threshold can bring disorders to sensitive individuals.     

The evaluation of endophyte toxin residues in sheep fat

Abstract

AIM: To monitor changes in concentrations of lolitrem B and epoxy-janthitrems in the fat of sheep grazing perennial ryegrass infected with wild-type- and AR37-endophyte, respectively, during the time of year when ryegrass staggers would be expected to be observed.

METHODS: Ten 5-month-old lambs with no previous exposure to endophytes were grazed on either wild-type (containing lolitrem B, n = 5) or AR37 (containing epoxy-janthitrems, n = 5) endophyte-infected perennial ryegrass (Lolium perenne L.) pastures between October 2008 and June 2009. Animals were regularly assessed for ryegrass staggers using the Keogh scale (0 = no signs, 5 = severe tremors). When a score of > 3.5 was observed animals were removed from the treatment pastures for 1 month. Fat biopsy samples were taken from each animal at approximately monthly intervals and analysed for endophyte metabolites using high performance liquid chromatography (HPLC) methods developed during this study. Regular herbage samples were also taken and concentrations of endophyte metabolites measured.

RESULTS: Efficient and reproducible methods to analyse both lolitrem B and epoxy-janthitrems in fat were developed. Concentrations of lolitrem B and epoxy-janthitrems in herbage and in sheep fat increased from late November to peak in mid-February. Ryegrass staggers was observed in both groups of sheep at this time. Following 1 month of grazing non-infected pasture mean concentrations in fat of lolitrem B decreased by 43% from 61.8 to 35.3 ppb, and of epoxy-janthitrems by 38% from 1032.0 to 639.5 ppb. Maximum concentrations in herbage of epoxy-janthitrems (35.7 ppm) were higher than of lolitrem B (3.4 ppm), but signs of staggers were less severe in sheep grazing pasture containing the former compared with the latter (median Keogh scores in late February were 2 and 3, respectively), consistent with epoxy-janthitrems being low potency toxins.

CONCLUSION: This study demonstrated that concentrations of epoxy-janthitrems and lolitrem B in sheep fat increased quickly during the initial phase of the study when concentrations in pasture increased, and decreased when animals were removed from pastures containing these compounds. These data will be used in the risk assessment of the endophyte metabolites.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

Cerebellar abiotrophy in a 6‐year‐old Arabian mare

Cerebellar abiotrophy (CA) is an uncommon neurological disease that most commonly affects Arabian horses. Affected horses are typically identified within the first 6 months of life. Intention tremor, wide based stance and ataxia are common clinical signs observed in affected individuals. No treatment is available for resolution of clinical signs. Definitive diagnosis is based on histopathological examination of cerebellar tissue, which is characterised by loss of Purkinje cell layer. This report describes a case of cerebellar abiotrophy that had a delayed diagnosis until 6 years of age.     

Polyneuropathy of Finnish horses characterised by inflammatory demyelination and intracisternal Schwann cell inclusions

REASON FOR PERFORMING STUDY: A neurological disorder characterised by pelvic limb metatarsophalangeal joint extensor paresis has been observed in numerous horses in Scandinavia for the last decade. Very little has been formally reported and there have been no detailed assessments of the neurological signs or neuropathological lesions. OBJECTIVES: To describe the epidemiological and pathological features of an outbreak of 'Scandinavian knuckling syndrome' in a riding stable in southern Finland. METHODS: Clinical neurological examination of 4 cases and neuropathological assessment of tissues of one case were performed. RESULTS: Eleven out of 17 horses fed on ryegrass from a common source showed progressive clinical signs of metatarsophalangeal extensor paresis necessitating euthanasia of 7 horses. Nervous system lesions in one horse consisted of a novel demyelinating, mildly inflammatory peripheral neuropathy, with BiP/GRP positive rough endoplasmatic reticulum Schwann cell inclusions. CONCLUSIONS: The clinical signs and lesions documented differ from any previously described equine polyneuropathy and suggest a primary Schwann cell lesion. POTENTIAL RELEVANCE: The classification of this disease as a novel demyelinating polyneuropathy may assist focused epidemiological investigations.     

Lead toxicosis in the horse: A review

Lead intoxication is rarely diagnosed in horses and can present a major challenge to the equine practitioner because of the variety of clinical signs. Horses with lead poisoning can develop gastrointestinal disturbances, neurological abnormalities, haematological changes, or nonspecific signs of weight loss, weakness and rough hair coat, which makes early diagnosis difficult. Fortunately, lead analysis of whole blood is routinely available and can confirm intoxication. Because of the well‐described lead‐induced peripheral neuropathies in horses, a thorough neurological examination is essential in the investigation of a suspect case. Once diagnosed, the source of lead has to be identified and further exposure prevented. Intoxication can be treated by administering chelating drugs and providing symptomatic and supportive care.     

Evidence that antibodies against recombinant SnSAG1 of Sarcocystis neurona merozoites are involved in infection and immunity in equine protozoal myeloencephalitis

Sarcocystis neurona is the principal etiologic agent of equine protozoal myeloencephalitis (EPM). An immunodominant protein of S. neurona, SnSAG-1, is expressed by the majority of S. neurona merozoites isolated from spinal tissues of horses diagnosed with EPM and may be a candidate for diagnostic tests and prophylaxis for EPM. Five horses were vaccinated with adjuvanted recombinant SnSAG1 (rSnSAG1) and 5 control (sham vaccinated) horses were vaccinated with adjuvant only. Serum was evaluated pre- and post-vaccination, prior to challenge, for antibodies against rSnSAG1 and inhibitory effects on the infectivity of S. neurona by an in vitro serum neutralization assay. The effect of vaccination with rSnSAG1 on in vivo infection by S. neurona was evaluated by challenging all the horses with S. neurona merozoites. Blinded daily examinations and 4 blinded neurological examinations were used to evaluate the presence of clinical signs of EPM. The 5 vaccinated horses developed serum and cerebrospinal fluid (CSF) titers of SnSAG1, detected by enzyme-linked immunosorbent assay (ELISA), post-vaccination. Post-vaccination serum from vaccinated horses was found to have an inhibitory effect on merozoites, demonstrated by in vitro bioassay. Following the challenge, the 5 control horses displayed clinical signs of EPM, including ataxia. While 4 of the 5 vaccinated horses did not become ataxic. One rSnSAG-1 vaccinated horse showed paresis in 1 limb with muscle atrophy. All horses showed mild, transient, cranial nerve deficits; however, disease did not progress to ataxia in rSnSAG-1 vaccinated horses. The study showed that vaccination with rSnSAG-1 produced antibodies in horses that neutralized merozoites when tested by in vitro culture and significantly reduced clinical signs demonstrated by in vivo challenge.     

Calculated forelimb flexor tendon forces in horses with experimentally induced superficial digital flexor tendinitis and the effects of application of heel wedges

OBJECTIVE: To calculate forces in the flexor tendons and the influence of heel wedges in affected and contralateral (compensating) forelimbs of horses with experimentally induced unilateral tendinitis of the superficial digital flexor (SDF) tendon. ANIMALS: 5 Warmblood horses. Procedure-Ground reaction force and kinematic data were obtained during a previous study while horses were trotting before and after induction of tendinitis in 1 forelimb SDF and after application of 6 degrees heel wedges to both forehooves. Forces in the SDF, deep digital flexor (DDF), and the suspensory ligament (SL) and strain in the accessory ligament (AL) of the DDF were calculated, using an in vitro model of the distal region of the forelimb. RESULTS: After induction of tendinitis, trotting speed slowed, and forces decreased in most tendons. In the affected limb, SL force decreased more than SDF and DDF forces. In the compensating limb, SDF force increased, and the other forces decreased. After application of heel wedges, SDF force in both limbs increased but not significantly. Furthermore, there was a decrease in DDF force and AL strain. CONCLUSIONS AND CLINICAL RELEVANCE: The increase in SDF force in the compensating forelimb of horses with unilateral SDF tendinitis may explain the high secondary injury rate in this tendon. The lack of decrease of SDF force in either limb after application of heel wedges suggests that heel wedges are not beneficial in horses with SDF tendinitis. Instead, heel wedges may exacerbate the existing lesion.     

Neurological disease associated with EHV-1-infection in a riding school: clinical and virological characteristics

An outbreak of neurological disease caused by EHV-1 infection is described with emphasis on diagnosis and prognosis for recumbent horses. In April 1995, an outbreak of the neurological form of Equine herpesvirus type 1 (EHV-1) occurred in a well-managed riding school with 41 horses: 34 horses showed a temperature spike and 20 some degree of neurological signs, of which 10 were nursed intensively in the indoor arena of the riding school for 3 to 20 days, 8 having to be maintained in slings for 2-18 days, while 9 needed bladder catheterisation b.i.d. for 2-16 days. Within the first 3 days, one horse was subjected to euthanasia and another horse died. Postmortem examination revealed a mild vasculitis with perivascular mononuclear cuffing and axonal degeneration in the central nervous system. Clinical diagnosis was confirmed by serology and virology: 28 horses seroconverted in one or more tests during the outbreak, whereas 12 had already high CF and SN titres in the first sample, suggestive of recent infection. Virus was isolated from nasal swabs of 4 horses, and identified as EHV-1 with type-specific monoclonal antibodies. Restriction enzyme analysis revealed that the EHV-1 strains from this outbreak belonged to genome type EHV-1.IP. The electropherotypes were identical to those from another, epidemiologically unrelated, outbreak of neurological disease 2 months earlier. The timing of the temperature spikes and seroconversions indicated that the infection was probably introduced by a horse purchased 3 weeks before neurological signs occurred. At follow-up one year later, the 10 horses that showed mild neurological signs had recovered completely. Of the 8 horses that survived intensive care, 3 had returned to around their former performance level (2 of which had been in slings), while the other 5 had become pasture-sound. At follow-up 4 years later, all pasture-sound horses had been subjected to euthanasia because of persistent mild ataxia and incontinence. In conclusion, the prognosis for recumbent horses due to EHV-1 infection is grave. For virological diagnosis, extensive and strategic sampling of febrile in-contact horses is required, and the EHV-1-specific glycoprotein G (gG) ELISA is a valuable tool for specific serological diagnosis of EHV-1 infection causing neurological disease.     

Molecular Investigation of the Viral Kinetics of Equine Herpesvirus‐1 in Blood and Nasal Secretions of Horses after Corticosteroid‐Induced Recrudescence of Latent Infection

Background: Recrudescence of latent equine herpesvirus 1 (EHV‐1) with subsequent viral shedding via nasal secretions is a potential source of infection for susceptible horses and has been implicated in outbreaks occurring in closed populations. Objectives: To describe the viral kinetics of reactivated EHV‐1 in blood and nasal secretions from latently infected horses after administration of corticosteroids, and to study the infectious nature of reactivated EHV‐1 to sentinel horses. Animals: Eight healthy horses. Methods: Four horses infected 4 months previously with EHV‐1 received dexamethasone on 5 consecutive days. Four seronegative horses served as sentinels and had direct contact with the latently infected horses. All horses were monitored daily for development of clinical signs. Whole blood and nasal secretions were collected daily for molecular detection and cell culture of EHV‐1. Serum was collected weekly for the detection of antibodies against EHV‐1. Results: All horses in the latently infected group showed transient molecular detection of EHV‐1 in blood and nasal secretions, but only 1 horse developed fever. Three latently infected horses developed an increase in antibody concentrations against EHV‐l. Viral cultures remained negative for all latently infected horses after corticosteroid administration. None of the sentinel horses developed clinical signs, viremia, viral shedding, or seroconversion. Conclusions and Clinical Importance: EHV‐1 was successfully reactivated after corticosteroid administration in latently infected horses. However, transmission of reactivated virus to sentinel horses was unsuccessful. Failure to effectively transmit EHV‐1 to susceptible horses may have resulted from the low level and short period of viral shedding in latently infected horses.     

Coma and respiratory failure due to moxidectin intoxication in a dog

Objective: To describe the clinical consequences following ingestion by a dog of a moxidectin‐containing equine deworming product. Few reports exist concerning the treatment and outcome of severe moxidectin toxicity. Treatment, known factors influencing intoxication, and prognosis are reviewed. Case summary: A 10‐month‐old female Border Collie ingested an unknown quantity of a moxidectin‐containing equine deworming product several hours before presentation. Severe neurological signs subsequently developed and included: ataxia, seizures, coma, and respiratory failure. The dog was treated with supportive care including intravenous fluids, activated charcoal, and positive pressure ventilation. Normal spontaneous respiration returned in 34 hours and the patient was discharged 58 hours after ingestion. Full recovery occurred within 1 week of intoxication. New information provided: This report describes moxidectin intoxication and associated respiratory failure in a dog that required mechanical ventilation. The dog's recovery was rapid. Despite severity of signs, the prognosis for patients with moxidectin intoxication is good with appropriate supportive care.     

Staggers induced by consumption of perennial ryegrass in cattle and sheep from northern California

Staggers was diagnosed in sheep and cattle from the northern California coast. The diagnosis was made on the basis of history of ingestion of perennial ryegrass (Lolium perenne) stubble, clinical signs of transient ataxia, which was aggravated by stimulation, and nearly complete recovery after removal of ryegrass as the primary forage. Morbidity was high, but death did not occur in any affected animals. The toxic endophyte, Acremonium lolii, was in most lower leaf sheaths from the ryegrass. Injection of extracts of the ryegrass from affected farms into mice induced signs of toxicosis. Additionally, ryegrass from all 3 farms contained the tremorgenic mycotoxin, lolitrem-B.