Edward, D. G. and Fitzgerald, W . A . (1951) - J . gen. Microbiol.

Huxtable, C. R. and Davis, P . E. (1976) - J . comp. Path. 86: 11. Kaklamanis, E., Thomas, L., Stavropoulos, K., Borman, I . and

Boshwitz, C. (1969) - Nature, Lond. 221: 860. Koshimuzu, K . and Ogata, M. (1974) - .lap. J . vet. Sci. 36: 391. Koshimuzu, K . , Yamamoto, K . and Ogata, M. (1973) - Jap. J .

vet. Sci. 35: 123. Rosendal, S. (1979) - in ‘The Mycoplasmas, vol. 2, - Human and

Animal Mycoplamas’, edited by Tully, J . G. and Whitcomb, R. F . , Academic Press, London, p 217.

Tully, J . G. and Rask-Neilsen, R. (1967) - Ann. N.Y. Acad. Sci. 143: 345.

(Accepted fo r publication 31 October 1984)

5: 566.

Intoxication of horses by lolitrem B in ryegrass seed cleanings

Mt. Pleasant Laboratories, B.L. MUNDAY Department of Agriculture, 1.M. MONKHOUSE P.O. Box 46, Launceston South, Tasmania 7249

Ruakura Animal Research Station, Private Bag, Hamilton, New Zealand

R.T. GALLAGHER

Neurotoxins called lolitrems have recently been isolated from perennial ryegrass (Lolium perenne L.) (Gallagher et a / 1981) and implicated as the principal causative neurotoxins of ryegrass staggers of livestock (Gallagher 1982). The major lolitrem neurotoxin, lolitrem B, has now been fully charac- terised (Gallagher et a / 1984). Outbreaks of ryegrass staggers occur most often after ingestion of ryegrass in intensive pasture grazing situations (Keogh 1973; Gallagher er a1 1977; Mortimer 1978), but ryegrass seed may contain enriched concentrations of the lolitrem neurotoxins which, if fed to stock, can cause a syndrome resembling ryegrass staggers (Gallagher et a1 1982).

This contribution reports the outbreak of a neurological syndrome in horses fed ryegrass seed cleanings, on a farm a t Legana, Tasmania. The horses were mixed age Welsh Mountain ponies, which were stabled without exercise, and fed exclusively on ryegrass seed cleanings. The seed cleanings were obtained from a seed processing establishment, which clearned seed harvested in northern Tasmania in the 19831 84 season. There were 2 main types of seed cleanings. One type was a “heavier”, “seedier” cleaning, which when fed to the horses was found to be more toxic than a “lighter”, “huskier” type. When the heavier seed cleanings were fed, signs appeared after 10 to 14 days. During this period. individual horses consumed 35 to 70 kg of cleanings.

The onset of the syndrome was sudden. The horses exhibited trembling, hypersensitivity to stimuli, abdominal muscular spasms accompanied by the passage of intestinal mucus and sometimes gave the impression of being diso- riented. The signs disappered within 2 to 3 days of cessation of feeding the seed cleanings. The syndrome was not produced i f the cleanings were fed to horses a t pasture, nor was it produced by feeding cocksfoot cleanings.

A sample of “heavy” ryegrass seed cleanings, which produced typical neurological signs in a 300 kg pony stallion, was analysed for lolitrems a t Ruakura Animal Research Station using a high pressure liquid chromatography method (Gallagher, Hawkes and Stewart, unpublished data). The cleanings contained 5.3 ppm of lolitrem B. Since i t has been shown that sheep fed ryegrass or ryegrass seed containing 2 ppm or more lolitrem B (Gallagher et a / 1982; unpublished) exhibit a syndrome resembling ryegrass staggers, the results of the analysis of the cleanings are consistent with the syndrome reported above in horses being due to intoxication with lolitrem neurotoxins. This conclusion is further sup- ported by the rapid disappearance of the signs, within 2 to

Australian Vererinary Journal, Vol. 6 2 , No. 6, June , 1985

3 days of cessation of feeding in the affected horses. The reversible nature of RGS neurotoxicity is well established, with affected animals normally recovering rapidly, that is within a few days of being transferred to safe pasture or being put on safe supplementary feed (Cunningham and Hartley 1959).

Although the syndrome produced by feeding the affected seed was similar t o naturally-octuring ryegrass staggers of horses (Cunningham and Hartley 1959; Munday and Morris 1978), the neurological signs were more intense and tenesmus has not previously been recorded in ryegrass staggers. These differences may well be due to the fact that the horses fed the seed being compelled to ingest significant quantities of toxin over a relatively short period, which could have resulted in a somewhat atypical clinical picture.

The authors acknowledge technical assistance with neuro- toxin analysis from Mr A.D. Hawkes and Miss J . Stewart.

References Cunningham, I.J. and Hartley, W.J . (1959) - N.Z. vet. J . 7: 1. Gallagher, R.T., Keogh, R.G., Latch, G.C.M. and Reid, C.S.W.

(1977) - N.Z. J . Agric. Res., 20: 431. Gallagher, R.T., White, E.P. and Mortimer, P.H. (1981) - N.Z.

vef. J . 29: 189. Gallagher, R.T. (1982) - Proc. XIII ISHAM Congr. edited by

Baxter, M., Massey Univ., Palmerston North, New Zealand. Gallagher, R.T., Campbell, A.G., Hawkes, A.D., Holland, P.T.,

McGaveston, D.A., Pansier, E.A. and Harvey, I.C. (1982) - N . Z . vet. J . , 30: 183.

Gallagher, R.T., Hawkes, A.D., Steyn, P.S. and Vleggar, R. (1984) - J . Chem. SOC. Chem. Commun. (Lond), p 614.

Keogh, R.G. (1973) - N.Z. J . exp. Agric., 1: S 5 . Mortimer, P.H. (1978) - Effects of Poisonous Plants on Livestock,

edited by van Kampen, R.F. and James, K.F., Academic Press, New York.

Munday, B.L. and Morris, D.1. (1978) - Tasmanian Plants Toxic for Animals, Tas. Govt. Printer, Hobart.

(Accepted for publication 2 December 1984)

An episode of acute selenium toxicity in a commercial piggery

Department of Agriculture, Victoria J . HILL* F. ALLISONt C. HALPINS

Selenium is an essential nutrient for livestock. In pigs, selenium deficiency has been associated with clinical disorders such as hepatosis dietetica and mulberry heart disease. Marginal deficiencies have been associated with illthrift and significantly reduced farrowing percentages (Underwood 1977).

O n the basis of this information selenium has recently become a standard feed additive for pigs’ rations. However, selenium is highly toxic and the safety margin between the therapeutic dose and the lethal dose is small. Animal requirements for selenium are approximately 0.05 mg/kg dry matter in the diet and the addition of 0.10 mg/kg (as the sodium salt) to feed for pigs has been approved by the United States Food and Drug Administration since 1974 (Ullrey 1980). However, supplemented rates of selenium in pig rations are sometimes much higher, up to 2 mg/kg (Costa 1984). It is generally accepted that dietary selenium levels as little as 5 mg/kg can result in growth inhibition and toxicity if intake is continuous, acute toxicity occurs a t levels greater than 20 mg/kg (Wilson et a/ 1983).

We investigated an outbreak of acute selenium toxicity in a commercial piggery associated with the incorrect formu- lation of a selenium supplemented diet. This episode repre-

Pakenham Street Echuca, Victoria 3625 t Regional Veterinary Laboratory, Bendigo, Victoria 3550 * “Attwood” Veterinary Research Labocatory, Mickleham

Road, Westmeadows, Victoria 3047

207

sents a timely reminder of the extreme caution required when handling and dispensing potentially toxic feed supplements.

The epizootic involved a commercial herd of 9,000 pigs in northern Victoria and occurred during October 1983. Within the piggery only 10 pens of grower pigs, totalling 256 pigs, were affected. The pigs were penned in age groups and were from 12 to 20 weeks old.

The common factor exclusive to these 10 pens was that they were all fed from the one silo. Feed delivery records to the piggery indicated that a 4 tonne batch of feed had been placed in that silo on Wednesday (day 0), although it was considered unlikely that the pigs received any of this feed prior t o Thursday afternoon (day 1) . N o other pigs in the piggery received this feed and n o other pigs developed clinical signs. Anorexia and depression were first noticed on day 2 and the first deaths occurred on day 3. After the initial investigation, a feed-borne toxin was implicated and the list of differential diagnoses included mycotoxins and arsenicals, or possibly selenium, copper or other heavy metals.

The incriminated feed was removed from consumption on day 3. However, new clinical cases continued to occur for a further 2 days. Between days 3 and 8, 42 pigs died or were killed for humane reasons. The attack rate for the epidemic was l 8 % , but this varied markedly between the age groups being 9 .5%, 35.2% and 1.6% for the 20-, 16- and 12-week age groups respectively. The case fatality rate was 89%.

On day 4 a check of dietary ingredients by the feed company established that sodium selenite concentrate had been added to the suspect feed at the rate of 300 mg/kg (300ppm). The level requested on the veterinarian’s prescrip- tion was 0.3 mg/kg. Discussions with the feed company revealed that the feed had been made up as a separate batch and could not have been sent to other piggeries. For the pigs which survived, the issues of meat inspection and public health standards concerninr: the tissue concentrations of selenium acceptable for hi:man consumption needed to be examined.

The clinical course of the intoxication began with depres- sion and anorexia 24 to 48h after the contaminated feed was received. A variety of clinical signs then developed appearing to involve the central nervous system. Mildly affected pigs developed a hind limb ataxia typified by walking upon the tip of the digits and with swaying of the hindquarters. More severely affected pigs were found in sternal recumbency but were able to rise to a “dog sitting” posture when goaded. Muscle fasciculation was evident in many of these pigs. Occasional vomiting was also observed but there was no diarrhoea. Pigs still more severely affected were in lateral recumbency and exhibited a generalised flaccid paralysis. There was no superficial pain response when tested by pin prick, or deep pain response to squeezing of the interdigital region. Most of these pigs soon became dyspnoeic and cyanosed, with a rapid weak pulse, and died I to 2h later. The cause of death was attributed to respiratory failure.

Five pigs in which clinical signs had progressed to sternal recumbency recovered over a period of 4 weeks. These pigs required force feeding initially because they were unable to drink or eat . N o other treatment was attempted. Pigs in the group which had not shown clinical signs of selenium toxicity remained anorectic for a further 3 to 4 days following removal of the suspect feed, after which appetite gradually returned to normal.

A necropsy performed on a moribund day 3 pig revealed a stark contrast between dehydrated carcase and oedematous viscera, of which the lung and pancreas were the most conspicuous. The lungs had not collapsed, the inter-lobular septa were distended by oedema, and froth was present in bronchi, trachea, nose and mouth. The liver was slightly pallid. Intestinal serosa was erythematous and the ingesta was dry and flecked with blood. Specimens, including feed, were gathered for histopathology, toxicology and bacteriol- ogy.

Histopathological examination of the liver showed the sinusoidal space to be diminished by enlarged hepatocytes and the kupffer and endothelial cell nucleii to be pyknotic. Myocardial degeneration and oedema were present in the heart. The gastric and duodenal mucosae were unremarkable, but the remainder of the intestines had congestion, focal erosions and luminad haemorrhages. The splanchnic connec- tive tissues and brain were variably oedematous and con- gested. Pathological examinations of this and later acute and chronic cases suggested a toxic microangiopathy. There were no significant bacterial growths. Ten surviving pigs slaugh- tered on day 35 had no signficant lesions in liver, kidney or muscle.

When the outbreak was first investigated samples of feed were collected for analysis, as were samples of tissue from 3 pigs which died after consuming the contaminated feed, and a comparable pig which had no access to the suspect feed. Further tissue samples were collected at day 15 from one clinically affected pig and a t day 35 from 10 remaining pen-mates to assess selenium residues a t slaughter.

Feed samples were dried under vacuum at - 1 8 T for 24 h, ground to pass a 2mm sieve, and digested in a mixture of nitric and perchloric acids. Selenium analyses were performed by the fluorimetric method of Watkinson (1966). Tissue samples were homogenised in 9 vols of double distilled water, and aliquots digested and analysed for selenium by the same method.

The contaminated feed sample was shown to contain 84.0 mg selenium per kg (dry matter basis) compared to 0.2 mg selenium per kg in the previous batch of feed from the same source.

Tisue selenium concentrations in clinically affected pigs were marginally higher than in the normal healthy pig which had not consumed the suspect feed (Table I ) , but were low in comparison to the concentrations recorded in weaner pigs with experimentally induced selenium toxicosis (Herigstad et a / 1973; Wilson e t a / 1983). At slaughter (day 3 9 , selenium concentrations were normal in muscle tissue but exceeded regulated standards f o r human consumption in kidney tissue. The dietary levels were so concentrated in this case that the pigs apparently either consumed the food and died before their tissues could accumulate significant concentrations of selenium or rejected the food completely. At more extreme dietary concentrations, a consistent clinical feature of sclen- ium toxicity is anorexia (Herigstad et a / 1973). No conclusion could be drawn from the variation in attack rates between age groups as this may have been due to either differences in the proportion of toxic and normal pellets received from the silo or differences in the severity of appetite depression.

Pathological lesions were essentially similar t o those of experimentally induced selenium toxicosis in weaner pigs (Herigstad et al 1973). Microangiopathy in the form of

T A B L E 1

Concentration of selenium in liver, kidney and muscle (mg/kg wet weight) at various intervals after delivery of feed suspected to have elevated levels of selenium

Animal s T i m e t Liver Kidney Muscle

Healthy (1)’ Day 5 0.75 2.00 0.36

Chronic c a s e (1) Day 15 1.55 3.00 0.48

* number of animals examined t t ime in days from delivery of suspect feed $ mean value - standard error of the mean

Acute c a s e s (3) Dat 3 to 5 2.90?0.63$ 3.77-tO.11 -

Survivors (10) Day 35 0.74rt0.07 2.38r0.08 0.22*0.01

208 Australian Veferinary Journal, Vol. 6 2 , No. 6 , June , 1985

capillary endothelial cell degeneration and necrosis apparently causes extensive interstitial oedema and acute hepatic and pneumonic incompetence leading to clinical manifestions of anorexia, muscular weakness, and primary and secondary central nervous system depression.

Supplementary selenium is stored in the kidney and liver and is rapidly excreted. Muscle tissue accumulates selenium only slowly. The Victorian Food and Drug Standards Regu- lations 1966 require that no food should contain more than 2 mg selenium/kg, on the basis of tissue selenium concen- trations a t day 35 (Table 1 ) the carcase muscle was accepted by the Victorian Meat Inspection Authority as being fit for human consumption, whearas all offal was rejected.

Many concentrated essential elements when given in excess are toxic to animals. In this case sodium selenite was added directly to the feed-stuff, a practice which may readily result in toxic overdoses when very small quantities of active ingredient are being supplemented. Such risks can be min- imised by using carrier compounds, commonly used in commercial rations.

The investigations were performed in co-operation with Dr E. Miles, Kyabram.

References Herigstad, R . R . , Whitehair , C. K . and Olsen, 0. E. (1973) - Am.

Ullrey, D. E. (1980) - J. Anim. Sci., 51: 645. Underwood. E. J . (1977) - Trace Elemenfs in Human a n d Animal

Watkinson, J . H . (1966) - Anal. Chem’ 38: 92. Wilson, T. M . , Scholz, R . W . and Drake, T . R . (1983) - Can. J .

(Accepted for publication 29 November 1984)

J . vct. Res. 34: 1227.

Nutrition, 4th ed, Academic Press, New York, p 302.

comp. Med. 47: 412.

Kangaroos and wallabies as carriers of Basidio bolus haptosporus

Graduate School of Tropical Veterinary Science, James Cook University of North Queensland, Townsville, Queensland 481 1

R. SPEARE

Oonoonba Veterinary Laboratory, Department of Primary Industries, Oonoonba, Townsville, Queensland 4812

A. D. THOMAS

The fungus Basidiobolus haptosporus is a cause of cuta- neous phycomycosis of horses in northern Australia (Connole 1973; Miller and Pot t 1980; Miller 1983). I t is a terrestrial fungus living in decaying vegetation (Drechsler 1947), but it has also been isolated overseas from the faeces and gut of amphibians, lizards and gekkos (Coremans-Pelseneer 1973; Porto and Milanez 1979; Gugnani and Okafor 1980) and in Australia from the faeces of the bearded dragon, Amphi- bolurus barbatus (Miller 1983). Miller (1983) proposed an epidemiological schema for equine basidiobolomycosis in which terrestrial lizards acted as reservoirs for the fungus, enabling it to survive adverse environmental conditions and suggested that infected lizards may also increase contami- nation of areas they frequented. Mammals have not been reported to act as reservoir hosts for B. haptosporus. This communication reports the isolation of Basidiobolus from the gastrointestinal tracts and faeces of macropods in northern Queensland.

Faecal material was obtained directly from the cloaca of live macropods or in the case of autopsied macropods, from the lower colon. Samples obtained a t autopsy and from the cloaca of live animals were collected using aseptic techniques. Samples of cleaned, artificial pouches in which joeys were restrained and of soil were also examined. The pouches were made of various types of cloth, were replaced when soiled and cleaned by laundering. The soil samples were taken from

Australian Veterinary Journal, Vol. 62, No. 6. June, 1985

the superficial layer, included detritus, and were collected from areas that had previously been frequented by joeys from which Basidiobolus had been isolated. All samples wee plated on 10% sheep blood agar and MacConkey agar plates, incubated in air a t 37°C and examined after 24 and 48 h. Fungal isolation was attempted on Sabourad dextrose agar* and Mycosel a g a r t with added thiamine, incubated a t 28°C for up to 14 days. Isolation of Basidiobolus from soil samples was attempted by plating directly as well as by the inverted culture technique of Coremans-Pelseneer (1973).

Some 285 of 480 specimens from 162 macropods were cultured for fungi. Seventeen isolates of Basidiobolus sp were recovered from 14 macropods: 15 from faeces, one from stomach contents and one from small intestinal contents. Basidiobolus sp was not isolated from 10 skin or hair specimens and 262 samples from various organs and other sites.

All the Basidiobolus sp grew on specific fungal isolation media a t 28°C and on the blood agar plates at 37°C. They produced thick walled chlamydospores occurring predomi- nantly in chains. Seven of the 17 isolates produced smooth- walled zygospores and using the criteria of King (1979) and

TABLE 1

Prevalences of Basidiobolus sp. in 16 species of macropod

Species Total No. of No. YO Joeys number joeys positive positive

Aepyprymnus rufescens r u f u s rat kangaroo 4

Lumholtz’ tree kangaroo 2

Hypsiprymnodon moschatus

m u s k y rat kangaroo 1

Lagorchestes conspicillatus

Dendrolagus lumholtzi

spectacled hare wallaby 5

agile wallaby 39

antilopine kangaroo 1

black stripe wallaby 10

Macropus agilis

Macropus antilopinus

Macropus dorsalis

Macropus giganteus eastern grey kangaroo - 60

Macropus parryi

Macropus robustus

Macropus rufus

Onychogalea fraenata

whiptail wallaby 4

eastern wallaroo 11

red kangaroo 7

bridled nailtail wallaby 2

Onychogalea unguifera northern nailtail wallaby 3

Pe troga le a ssim ilis Palm Island rock wallaby 6

purple neck Pe trog a le p urp u reicollis

rock wallaby 1 Wallabia bicolor

2

0

0

1

33

0

4

48

3

4

7

0

3

3

0

A

0

0

0

1

1

0

0

6

0

2

1

0

2

0

0

1

0

0

0

100

3.0

0

0

12.5

0

50

14.3

0

66.7

0

0

25 swamp wallaby 6 Total 162 112 14 12.5

Oxoid Australia Ltd, Heidelberg West, Victoria t BBL, A . E . Stansen and Co Pty Ltd, Mt Waverley, Victoria

209