Clinical communication — Kliniese meededeling

Tremorgenic neuromycotoxicosis in 2 dogs ascribed to the ingestion of and possibly roquefortine in rice contaminated with crustosum

T W Naudéa, O M O'Brienb, T Rundbergetc, A D G McGregord, C Rouxb and A Flåøyenc

of 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, acts by interfering with the but contained 2.6 µg/g of the 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 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 , 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 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::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). This owner’s 2nd dog, a 3-year-old cross-bred (100 m , followed by 50 m ). The extracts could, however, not be confirmed by Schnauzer (mass c. 25 kg), was also shiv- were filtered (Whatman No. 1), com- 1 Arnold et al. who found the LD50 ranging ering and exhibiting loss of balance. He bined, and evaporated to dryness under from 169–189 mg/kg in male and female gave the dog some home medication by reduced pressure at 50 °C on a rotatory Swiss-Webster and C17 mice. The signs mouth which induced vomition. This dog evaporator. The residue was taken up in of intoxication were hypokinesia and was then, 2 hours after the 1st one, also methanol (5 m ), and a portion of the standing on their hind legs for prolonged taken to the consulting room and pre- methanol solution (0.5 m ) was run onto a periods of time and eventually, in those sented with slight salivation and muscle Bond Elute C18 cartridge (500 mg) (Varian, that were dying, quiescence, adipsia tremors. It was similarly treated with atro- USA) which was then successively eluted and anorexia No tremorgenic signs were pine, clanobutin and xylazine and also with 1 m methanol:water (1:1, v/v) and reported. Roquefortine and penitrem A hospitalised. 2m methanol. Prior to LC-MS analysis, were isolated from cultures of Penicillium Both dogs were clinically normal the the methanol:water and methanol frac- commune obtained from mouldy cotton- next morning and were discharged. tions were filtered using a Spin-X seed meal during biological trials using The owner’s son fortunately collected (Corning, New York) centrifugal filter. in day-old cockerels to vomitus from the 1st dog off the floor and LC-MS and LC-MS-MS analyses. Chro- monitor the isolation38. No dosages are kept it in their refrigerator in a tightly matography was performed on a Waters given but the fractions containing the 2 closed wide-mouth jar for c. 1 week Symmetry C18 column (5 µm, 4.6 × individual were differenti- when, at our request, it was transferred to 150 mm) (Milford, USA), using a TSP ated by the roquefortine resulting in the veterinary consulting room where it P4000 pump and an AS3000 autosampler paralysis while the penitrem A resulted in was kept frozen. At this stage a clearly vis- (San Jose, USA). A gradient mobile phase tremors. Although roquefortine has been ible green fungal covering was present on consisting of mixtures of methanol and implicated in severe excitatory neuro- the outer surface of the rice grains in the 0.1 % HCOOH and 0.01 M ammonium toxicity of dogs, resembling strychnine bottle. The vomitus was kept in a frozen acetate was used. The methanol level of poisoning18,19,31, no experimental data on state up to the stage that the mycological the mobile phase, starting at 25 % (2 min its toxicity in dogs could be traced. investigation and, as far as practicable initial hold) was raised to 60 % over Roquefortine and penitrem A are pro- also the chemical analyses, were carried 15 min, then to 95 % over 5 min, and held duced concurrently by Penicillium crus- out. for 10 min. The flow rate was 0.7 m /min

212 0038-2809 Tydskr.S.Afr.vet.Ver. (2002) 73(4): 211–215 the m/z 390 (MH+) ion of roquefortine fragmented to afford m/z 322 (loss of

imidazole C3H4N2), and 193 ions (Fig. 4a), whereas the m/z 634 (MH+) ion of penitrem A fragmented to afford m/z 616 and 558 ions (Fig. 4b), corresponding to the consequential loss of water and the elements of acetone, respectively.The loss of a mole of acetone is of particular diagnostic significance in that it can be attributed to the loss of the bridging

–C(CH3)2–O– ether linkage present in penitrem type mycotoxins produced by Penicillium species. The MS-MS spectra of the m/z 390 and 634 ions, shown in Fig 4a,b, corresponds with those of au- thentic specimens of and penitrem A, respectively. Quantification, performed in MS-MS mode, demonstrated the presence in the Fig. 3: (a) LC-MS and (b) LC-MS-MS chromatograms of the methanol fraction of the vomitus extract. vomitus of 34 µg/g wet mass of roque- fortine C (sum of methanol and metha- and 20 µ of the filtered methanol-water, methanol–water and the methanol nol-water fractions contributions) and or methanol fractions were injected. fraction. Penitrem A (m/z 634, MH+) was 2.6 µg/g wet mass of penitrem A. The HPLC system was coupled to a only detected in the methanol fraction. Finnigan MAT, LCQ ion trap mass spec- These mycotoxins were initially identi- Analysis for other possible neurotoxic trometer operated with an atmospheric fied on the basis of their MH+ ions and re- substances pressure chemical ionisation (APCI) tention time comparison with authentic Analysis of the remainder of the vomitus interface (San Jose, USA). Spectral data specimens (Fig. 3a, peaks at 14.5 and at (11 g) for organochlorine, organophos- were acquired in both MS in full scan and 25.3 min). Protonated molecular ions phor and carbamate insecticides by MS-MS in full scan positive ion modes. corresponding to penitrems B, C, D, E or F GC/MS at the Veterinary Institute, Oslo, The MS ion injection time was set to could not be detected in either of the and (7 g) for strychnine by TLC at the Di- 400 ms with a total of 2 micro-scans per fractions of the sample. vision of Toxicology at the Onderstepoort second. A vaporisation temperature at Acquisition of mass spectral data in Veterinary Institute, Pretoria, proved to 350 °C, a sheath gas rate at 25 units nitro- MS-MS mode greatly increased the selec- be negative. gen, a corona discharge voltage of 4.5 V tivity and signal to noise ratio (and hence and a capillary temperature of 200 °C the detection limits) of the analytical DISCUSSION were used. procedure (see Fig. 3b). Ions at m/z 390 In South Africa the usual toxicological and 634, corresponding to the MH+ ions differential diagnoses in dogs considered Results of roquefortine C and penitrem A, respec- and tested for when agitation, trembling LC-MS analyses, performed in full scan tively, were fragmented and analysed in and nervous stimulation eventually mode, demonstrated the presence of the MS-MS stage. Under the MS-MS progressing to convulsive seizures, roquefortine C (m/z 390, MH+) in both the conditions applied in this investigation, prostration and death are encountered, are (in sequence of importance) poison- ing by of the organochlorine, organophosphor or carbamate groups and secondly, strychnine. Lately fluoro- acetate (illegally obtained and used) has been added as poisoning does occur and a practicable test for it has been developed22. Lead poisoning is rare but is occasionally encountered. Metaldehyde poisoning, even more rarely encountered here, must also be considered (T W Naudé, pers. obs., 2001). The oral toxic dose of penitrem A for the dog is unknown (vide supra). Unfortu- nately it was also impossible to arrive at a reasonable estimate of what dose had resulted in intoxication in this particular instance. Only the amount of penitrem A in the a specimen of the 1st dog’s vomitus, which had been kept in the owner’s refrigerator for a week before it was frozen and finally analysed, is known. Fig. 4: MS-MS spectra of (a) roquefortine C and (b) penitrem A. Other unknown factors are the amounts

0038-2809 Jl S.Afr.vet.Ass. (2002) 73(4): 211–215 213 of infected rice actually eaten by each dog and quiescence occurred at a very much recovery from the latter more sedation is and disgorged later. Finally the amount of higher LD1. In fact Wagener et al.38, using required, xylazine (which was used with toxin that could have been produced dur- day-old cockerels to monitor their isola- success) seems indicated. It is of interest to ing the c. 1-week storage of the vomitus in tion, clearly differentiated penitrem A note that Lowes et al.19 found the tremor- the same fridge where the mycotoxins from roquefortine on grounds of the genic state they had ascribed to roque- had initially been produced on rice, has to tremorgenic activity of the former in con- fortine poisoning totally unresponsive to be considered. Nevertheless in our opin- trast to paralytic action of the latter. diazepam. Peterson et al.26 also found that ion a diagnosis of penitrem A poisoning Yamazaki (1980)41 groups roquefortine diazepam to some extent controlled the (possibly influenced by the presence of chemically with fumitremorgen and clinical signs of the tremorgen verru- roquefortine) is justified. Consequently, verruculogen but points out that it differs culogen but did not abolish it as com- this should now be added to the list of structurally from these 2 by having an pared to the barbiturates. It seems possible neurotoxicities of dogs in South isoprene unit (in a novel arrangement as a probable that this could be the same with Africa. Fortunately, suitable qualitative 1,1-dimethylallyl group) on position 3 of penitrem A. The use of activated charcoal and semi-quantitative analytical TLC the indole nucleus in stead of the usual 2 per os, as in most poisonings, is clearly techniques for penitrem A20 and position (see arrow in Fig. 2). indicated. In view of the fact that liver roquefortine34 are available and could be In Canada, roquefortine was present in damage was reported in dogs with used routinely in a diagnostic facility. the stomach contents of a significant penitrem A intoxication13, liver support- The air samples from the refrigerators as number of dogs that had died of or had ive therapy should also be considered. well as the kitchen were free of P. crus- exhibited signs resembling strychnine The scavenging nature of dogs and their tosum. Although the owner of the dogs poisoning but where strychnine tests interest in and occasional predilection for had stated that they had earlier kept some were negative. The toxicity was ascribed garbage and foul smelling food8 probably pears in the kitchen and the fungus is to roquefortine although quantification make this the species most prone to known to be weak pathogen of, amongst was not done and it was admitted that no penitrem A poisoning, particularly as others, pomaceous fruit29 , this could not reports on the clinical signs of this intoxi- P. crustosum is an ubiquitous spoilage be related to the origin of the contamina- cation in the dog were available19,31. organism of food25,29,39,40. The reason why tion. Although penitrem A is mentioned in this intoxication has not been diagnosed The refrigerator in which the incrimi- these publications, it was not excluded in previously in South Africa is probably nated rice infection had developed was at either case. Braselton and Rumler5 re- that veterinarians had been unaware of a maximum temperature of 3 °C and a ported the presence of roquefortine in the this neuromycotoxicosis and had not minimum of 1 °C. One of the diagnostic stomach content of 7 dogs that had been considered it. characteristics within the subgenus Peni- tested for strychnine with negative re- cillium is the ability of these species to ger- sults. However, significantly, in 6 of these ACKNOWLEDGEMENTS minate and grow at low temperatures23,28 cases where sufficient sample were We thank the Veterinary Institute, Oslo, and produce toxins28. It was demon- available, penitrem A was also present Norway for the analysis of the strated that P. crustosum grown at 4 °C on and they were the first to speculate on the vomitus, and the Division of Toxicology rice produced considerable amounts of possible synergistic action of these 2 of the Onderstepoort Veterinary Institute penitrem A, which peaked at 50–85 days . Quantification was, however, for testing for strychnine. Critical evalua- with a high level still being present at unfortunately again not reported. In a tion of the manuscript by Professors A 120 days. By contrast, at 20 °C, peak pro- further 34 out of 37 specimens ranging Wilkins of Agresearch, Ruakura, New duction of toxin on this substrate was at from stomach contents and vomitus to Zealand, and R Vleggaar of the Organic 10–25 days and by 50 days the level had mouldy food, the presence of both toxins Chemistry Department, University of been depleted greatly16. This particular were confirmed by MS-MS and in the Pretoria, is gratefully acknowledged. We isolate also proved to produce penitrem A other 3 the suspected presence of peni- also thank the owner’s son, Mr Klaus when grown on rice at 10 °C (T Rund- trem A was too low to confirm by MS-MS Schwerdtfeger of Pretoria, for collecting berget, Veterinary Institute, Oslo, pers. (E Braselton, Michigan State University, and storing the vomitus for us. comm., 2001). pers. comm., 2000). Penitrem A intoxication in dogs has There appear to be little justification in REFERENCES clearly been established by administra- diagnosing roquefortine poisoning in 1. Arnold D L, Scott P M, McGuire P F,Harwig tion of purified toxin13 and the signs of dogs. There are no data on its toxicity in J, Nera E A 1978 Acute toxicity studies on roquefortine and PR toxin, metabolites of intoxication are indistinguishable from the dog, no definite data confirming its in the mouse. Food and those in field cases where it occurs con- tremorgenic potential (in fact it rather Cosmetic Toxicology 16: 369–371 currently with roquefortine. In the 3 natu- appears to have a paralytic action) and it 2. Arp L H, Richard J L 1979. Intoxication of ral outbreaks of intoxication ascribed to has a relatively low toxicity in mice. It dogs with the mycotoxin penitrem A. 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