Clinical communication — Kliniese meededeling Tremorgenic neuromycotoxicosis in 2 dogs ascribed to the ingestion of penitrem A and possibly roquefortine in rice contaminated with Penicillium crustosum T W Naudéa, O M O'Brienb, T Rundbergetc, A D G McGregord, C Rouxb and A Flåøyenc of strychnine in that it inhibits the inhibi- ABSTRACT tory neurotransmitter glycine in the Two dogs developed alarming tremorgenic nervous stimulation shortly after ingesting central nervous system. In particular the discarded rice that had been forgotten in a refrigerator for an undetermined period and glycine concentration in the brain is that was covered with a grey-green mould. Both dogs exhibited vomition followed by slight lowered and the tremors can be abolished salivation, tremors and ataxia and 1 showed such severe agitation and seizures that it by mephenesin or nalorphine that raises necessitated anaesthesia with thiopentone followed, on recovery, by xylazine. The other the glycine level. The tremors are thus dog was only sedated with xylazine. They made an uneventful recovery. The rice vomitus most probably of supraspinal origin. yielded a pure culture of Penicillium crustosum. On chemical analysis it was negative for Later Norris et al.24 concluded that the organochlorine, organophosphor and carbamate insecticides, as well as for strychnine, mycotoxin acts by interfering with the but contained 2.6 µg/g of the mycotoxins penitrem A as well as 34 µg/g of roquefortine as glutamate, aspartate and GABA amino determined by LC-MS and confirmed by MS-MS. This is the 1st South African case of naturally occurring penitrem A toxicosis and also the 1st case where quantification of the acid neurotransmitter release mecha- levels of mycotoxins in dog vomitus is reported. The tremorgenicity of roquefortine and its nisms. This is expected to result in contribution towards this syndrome, is questioned. anomalous release of both inhibitory and excitatory transmitters at central and Key words: dogs, mouldy rice, neuromycotoxicosis, Penicillium crustosum, penitrem A, roquefortine, tremorgen. peripheral synapses and the loss of the neural coordination controlling muscle Naudé T W,O'Brien O M, Rundberget T,McGregor A D G, Roux C, Flåøyen A Tremorgenic action. neuromycotoxicosis in 2 dogs ascribed to the ingestion of penitrem A and possibly The i.p. LD of penitrem A for mice is roquefortine in rice contaminated with Penicillium crustosum. Journal of the South African 50 1.05 mg/kg15 whereas dogs died at i.p. Veterinary Association (2002) 73(4): 211–215 (En.). Department of Paraclinical Sciences, 13 Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110 doses of 0.5 mg/kg and higher . In addi- South Africa. tion to the above 2 species, rats, rabbits, guinea-pigs, hamsters, chickens, calves, sheep and swine are susceptible to this neurotoxic substance9,10. The signs of INTRODUCTION is well known. The fact that certain of intoxication are similar in all species and Penicillium crustosum Thom occurs in these fungi may produce toxic metabo- set in within 5–10 min after i.p. or p.o. the air and soil and on various substrates lites such as the alkaloid roquefortine and administration. Sustained tremors, ataxia such as food, meat products and feed. In PR toxin12 is, however, not common and muscular rigidity progress in a addition is known as a weak pathogen of knowledge. dose-related fashion to fatal clonic or pomaceous and citrus fruit as well as In man, eating of grossly overripe or tetanic convulsions. Agitation, hyper- cucurbits29, but it is also known as an ubiq- mouldy cheese or other severely mouldy excitability and a change in temperament uitous spoilage organism that may cause food hardly ever occurs because of its may be seen. In dogs, vomition often post-harvest rot of fruit. It also commonly offensive smell, taste and nature. Dogs occurs and may be life-saving. An un- contaminates foodstuffs which may then are, however, not as discriminating and usual feature of this neurotoxin is that it is become toxic16,28,32. Strains within this toxicity due to ingestion of such products also hepatotoxic resulting in dose-related species are known to produce several sec- is on record19,31,32. centrilobular hepatic haemorrhage and ondary metabolites that act as mycotoxins, Penitrem A (Fig. 1) is one of a group of necrosis in dogs and fatty changes in including the tremorgens penitrem A–F 6 related penitrems and is closely-related calves with concurrent elevation of serum as well as the mildly neurotoxic roque- to the tremorgenic paspalines produced aspartate transaminase activity. Owing to fortine11,17,21. by Claviceps paspali on Paspalum spp., the tremors a sharp rise in creatine kinase The utilisation of different Penicillium the cause of paspalum staggers20. This activity also occurs. Recovery from intoxi- spp. (e.g. P.roqueforti and P.camembertii) for tremorgenic mycotoxin has a single nitro- cation is usually complete and without the production of specific types of cheese gen atom in a substituted indole moiety sequelae. Poisoning in dogs due to inges- and a multi-ring structure derived from tion of P. crustosum-contaminated cream a Department of Paraclinical Sciences, Faculty of 2 33 Veterinary Science, University of Pretoria, Private Bag mevalonate units. It is produced by vari- cheese , mouldy walnuts and a severely X04, Onderstepoort, 0110 South Africa. ous Penicillium spp. but most commonly mouldy hamburger bun4 has been as- bBiosystematics Division, ARC–Plant Protection Re- 3 search Institute, Private Bag X134, Pretoria, 0001 South by P. crustosum isolates . By common cribed to penitrem A. The signs of intoxi- Africa. agreement the trivial name tremortin A cation were indistinguishable from cVeterinary Institute, Oslo, Norway. earlier in use for the toxin, was replaced those described with the purified toxin13. d78 Alcade Rd, Lynnwood Glen, Pretoria (Deceased). with penitrem A28. No data could, however, be traced on *Author for correspondence. 6 E-mail: [email protected] atovi et al. regarded the mechanism the oral toxic dose of penitrem A to the Received: May 2002. Accepted: August 2002. of action to be in some way similar to that dog. 0038-2809 Jl S.Afr.vet.Ass. (2002) 73(4): 211–215 211 tosum21 but no studies have been done on RESULTS the effects of the concurrent exposure to and possible synergistic effects of the 2 Mycological investigation toxins5. A pure culture of a fungus was isolated Although penitrem A/roquefortine from the vomitus, then inoculated and toxicity in dogs has been recorded as early grown according to the regime of Pitt28. as19792 and 198831, respectively, this is the This briefly consists of point-inoculating 3 1st report of this intoxication in South Petri dishes of Czapek yeast autolysate Africa. agar (CYA)27 and 1 Petri dish each of malt extract agar (MEA)4,28 and 25 % glycerol ni- trate agar (G25N)27. The MEA, G25N and Fig. 1: Structural formula of penitrem A. CASE REPORT One evening a dog owner discarded the 1 CYA plate were incubated at 25 °C, and contents of a plastic-covered bowl of the 2 remaining CYA plates at 37 °C and cooked rice which had been forgotten at 5 °C, respectively. Macroscopic and micro- the bottom in his refrigerator for an scopical morphology on all media was unknown period and which had become examined after 7 days’ incubation. The overgrown with a dark-green mould isolate was identified as P. crustosum layer, on his compost heap in the garden. Thom, a terverticilate Penicillium in the His 2 dogs discovered it and ate it within subgenus Penicillium, series Viridicata an hour. About 2 h later his 2-year-old, Raper & Thom ex Pitt28,37,40. spayed Miniature Schnauzer bitch (mass Petri dishes with potato carrot agar and c. 20 kg) startedshivering violently and 1.5 % malt extract agar, respectively, were Fig. 2: Structural formula of roquefortine. vomited repeatedly, disgorging a rice-like exposed to the air for 20 minutes in the Note (arrow) the isoprene unit (in a novel substance. He took her to his veterinarian kitchen/scullery and the 2 refrigerators at arrangement as a 1,1-dimethylallyl group) on where the dog presented with ataxia, the owner’s home to sample for fungal position 3 of the indole nucleus. muscle tremors, polypnoea, severe agita- spores. Fungal colonies which developed tion and generalised seizures. The dog on incubation at 25 °C were identified. Roquefortine (a diketopiperazine) is a was anaesthetised with thiopentone Mycoflora of the kitchen yielded only the 5-nitrogen-containing molecule (Fig. 2). It (Intraval, Rhône-Poulenc) i.v. (25 mg/kg), usual Cladosporium spp. dominated range is a stable metabolite produced by P. treated s.c. with atropine sulphate (Cen- of fungi one would expect. roqueforti, P. crustosum and P. chryso- taur Labs) (0.1 mg/kg) and clanobutin genum30. It has been isolated from pro- (Bykahepar, Centaur Labs) (27 mg/kg) Chemical investigations cessed cheese35 and has even been and hospitalised. Two hours later it was implicated in human intoxication from sitting up in its cage but was still exhibit- Analysis for mycotoxins mouldy beer invaded by P. crustosum7. ing muscle tremors and slight ataxia. Sample treatment. Vomitus(10 g wet mass) The i.p. LD50 first reported in mice was Xylazine (Rompun, Bayer) (10 mg/kg) was introduced into a 500-m stoppered 15–20 mg/kg with opisthotonoid seizures was given i.m. to sedate the dog over- flask and shaken up twice with dichloro- elicited by external stimuli during pros- night. methane:methanol:ethyl acetate (2:1:3, tration and atonic posture (Fraysinnet On returning home c. 1 hour later the v/v/v) containing 0.1 % formic acid and Frayssinet cited in Scott et al.36).