dicate that although BEF infection occurs in buffalo, the mor-
bidity rate is lower than for cattle and the virus is less
pathogenic. Reports of buffalo infected in the field tend to
support these results. A widespread BEF epidemic which
swept through south east China in September-October 1987
caused severe disease with 80 to 90% of the cattle in each herd
being affected (Chang Zeji, personal communication).
However, the prevalence of disease in buffaloes was con-
sidered to be much lower and the clinical signs were milder.
The results reported here are in contrast to the experiments
described by Young (1979) in which clinical disease could not
be induced in 3 buffaloes infected with BEF. The reasons for
these differences are not known although differences in virus
strain or infectivity may play a role.
We wish to thank Mr Huang Junyao, Director, Foshan City
Animal Husbandry Bureau and his staff for the use of their
facilities. This work was partly supported by the Australian
Centre for International Agricultural Research, Project No
8455.
References
Bai W , Tian F, Wang C, Jiang C and Zhang Z (1986) - Proc 4th
Symp Arbo Res Aust. Ed by TD St George, BH Kay and J Blok, p
279
Cybinski DH and Card G P (1986) - Proc 4th Symp Arbo Res Aust.
Ed by TD St George, BH Kay and J Blok, p 289
-
Cybinski DH, St George TD and Paul1 NJ (1978) Aust Vet J 5 4 1
Mackerras IM, Mackerras MJ and Burnet FM (1940) - Bull Counc
Sci Ind Res 136
Malviya HK and Prasad J (1977) - Indian Vet J 54: 440
Mohan RN (1968) - Vet Bull 38: 567
St George TD, Standfast HA and Cybinski DH (1978) - Aust Vet J
54: 558
St George TD (1981) - In: Virus Diseases of Food Animals a World
Geography of Epidemiology and Control Ed by Gibbs EPJ
Academic Press, London, p 541
Tian F, Jiang C, Zakrzewski H and Davis SS (1987) - Aust Vet J 6 4
159
Uren MF and Murphy GM (1985) - Vet Microbiol 10: 505
Young P (1979) - Aust Vet J 55: 349
(Accepted for publication 14 June 1989)
The toxicity for cattle of bufadienolide cardiac
glycosides from Bryophylrurn tubiforurn flowers
Queensland Department of RA McKENZlE
Primary Industries, the late FP FRANKE
Animal Research Institute, P J DUNSTER
Yeerongpilly, Queensland 4105
Three bufadienolide cardiac glycosides, named bryotoxins
A, B and C, occur in the flowers of the poisonous plant
Bryophyllum tubiflorum (mother-of-millions) (Capon et al
1985, 1986). These bryotoxins also occur in flowers of other
species of Bryophyllum and in leaves, stems and roots of these
plants (McKenzie et a1 1987). The syndrome resulting from in-
gestion of B. tubiflorum by cattle is consistent with cardiac
glycoside poisoning (McKenzie and Dunster 1986). However,
it is unclear if the diarrhoea and omasal lesions noted by
McKenzie and Dunster (1986) are also due to cardiac
glycosides or whether there are other toxins causing these ef-
fects. Consequently we sought to determine if the known
bryotoxins from B. tubiflorum were capable of reproducing
the effects produced by the plant itself. If these compounds
were shown to account for the disease, the need for further
searching for toxins and the subsequent animal experiments
would be reduced o r removed.
B. tubiflorum flower heads collected in 1985 in the Moreton
region of south-eastern Queensland (Queensland Herbarium
Voucher BRI 361717) were frozen and stored at -2OOC.
Frozen plant material ( 2 kg in 200 g lots) was macerated for 5
min in a blender with 50% aqueous ethanol (10 x 1 litre). The
resulting slurry was filtered with suction and the filtrate con-
centrated under vacuum to about 500 ml. Repeated extraction
374
of the aqueous concentrate with 6 portions of ethylacetate
(each 500 ml) and concentration of the combined organic ex-
tracts gave a dark-reddish-coloured gum (4 g). The latter was
dissolved in 50% aqueous ethanol and applied, in 5 lots, to a
fractionating column (30 cm length, 50.8 mm diameter) filled
with reverse phase silica gel'.
The components of the mixture were eluted from the col-
umn with 40% aqueous methanol at approximately 7 ml/min.
The eluate was continuously monitored with a refractive index
detector. Bryotoxin A eluted from the column at approximate-
ly 800 to 1100 ml. Concentration of this fraction under reduc-
ed pressure from the 5 lots gave 341 mg (0.017% of the star-
ting mass) of pure bryotoxin A, identical in all respects by
chromatographic (thin layer chromatography on silica gel and
reverse phase silica gel) and spectroscopical (proton nuclear
magnetic resonance) comparisons with authentic material sup-
plied by RJ Capon, Australian National University.
The preparation of bryotoxins B and C (as a non-separable
mixture) required 2 chromatographic steps. Extraction with
aqueous ethanol, filtration, concentration and extraction into
ethylacetate were carried out as described above. The residue
was dissolved in 60% aqueous methanol and applied, in 5 lots,
to the reverse phase column. The components of the mixture
were eluted with 60% aqueous methanol. The first 800 ml
were discarded and the following 500 ml collected, which con-
tained all of the bryotoxins B and C. This fraction was concen-
trated under reduced pressure and the residue (491 mg from 5
lots) dissolved in ethylacetate and chromatographed on silica
gel 60 (70-230 mesh, 32 cm length, 40 mm diameter) with
ethylacetate as eluent. Fractions, each of 10 ml,were collected
and checked for content and purity of bryotoxins B and C by
high performance chromatography. Fractions containing
these were combined and concentrated yielding 103 mg
(0.005vo of the starting mass). Identity with authentic
bryotoxins B and C was again established by chromatographic
and spectroscopic comparisons.
Four weaned calves, 3 females and 1 male, 3 to 4 months old
and with a mean weight of 74 kg (range 64 to 83 kg) were used
in a dosage experiment in accordance with national guidelines
for the care and use of experimental animals (Anon 1985). The
techniques of husbandry, clinical examination, heart rate
monitoring, electrocardiography, clinical pathology, necropsy
and histopathology employed were those used previously
(McKenzie and Dunster 1986, 1987).
The calves were injected through the left flank into the
rumen. For dosing with bryotoxin A , 250 mg was dissolved in
6 ml ethanol and injected into a female calf weighing 64 kg
(3.9 mg/kg). A male control was injected with 6 ml ethanol
alone. For dosing with bryotoxins B and C combined, 280 mg
was dissolved in 1 ml dimethylsulphoxide (DMSO) and in-
jected into a female calf weighing 73 kg (3.8 mg/kg). A female
control was injected with 1 ml DMSO alone. DMSO was used
as the solvent for bryotoxins B and C because of their limited
solubility in ethanol. The doses of bryotoxins used were
estimated from the data of McKenzie and Dunster (1986) and
McKenzie et al (1987) to be those likely to be present in a
fatally-toxic dose of whole B. tubiforurn.
The calf given bryotoxin A became lethargic and developed
inappetence, ruminal atony and diarrhoea by 10 h after dos-
ing. These signs persisted for the rest of the experiment. The
faeces were mucoid with occasional flecks of blood. From 3
days after dosing, both heart block, which was manifested by
missing beats on auscultation, and a jugular pulse were noted
occasionally. On the tenth day after dosing, drooling of saliva,
tachycardia and dyspnoea occurred. The calf was weak and
very lethargic and was killed with intravenous pentabarbitone.
Changes noted in electrocardiograms with time after dosing
were lengthened PR intervals (first degree atrioventricular
block), shortened QT intervals (Figure l ) , reversed T wave
polarity and depressed S T segments (both persistent from day
4 onwards) and intermittent second degree atrioventricular
blocks (types 1 and 11) on days 3 , 4 , 7 , 8 and 9. Plasma concen-
~~ ~
Matrex@silica C18,6nm pore diameter, 35-70 pm particle size,
Amicon Corporation, Danvers, Massachusetts, USA
Australian Veterinary Journal. Vol. 66, No. 1 I , November, 1989
 [rations of urea and creatinine increased steadily and those of lace 1 2
calcium decreased steadily during the experiment (Figure 2).
Plasma concentrations of bilirubin increased erratically
(Figure 2). Plasma activities of glutamic oxaloacetic tran- P
saminase and creatinine phosphokinase and plasma concen-
trations of glucose increased slightly above normal limits only
on the tenth day after dosing. The values of other plasma con-
B
stituents remained within normal limits.
At necropsy, subendocardial haemorrhages were seen over
the papillary muscles of the left ventricle. There was extensive p-d
ulceration of the edges and surfaces of the ornasal leaves.
Many ulcers had perforated the leaves completely. Some con-
gestion and petechiae occurred in the abomasal fundus. A few
atelectatic lobules were noted in each lobe of the lungs. The
liver appeared slightly orange in colour and the bile was viscid
and with a gritty texture. Histological findings were scattered
foci of degeneration and necrosis in the myocardium of both
ventricles and the septum accompanied by swollen vascular
endothelial cells in the left ventricle, fatty change and swelling
of hepatocytes which was more pronounced towards the
periphery of acini and a few scattered accumulations of bile in
canaliculi. No renal lesions were detected.
The calf given bryotoxins B and C combined became restless
and stopped feeding within 1 h after the injection. Its ruminal
movements were weak with fewer than one contraction noted
every 2 min. By 2 h, abdominal pain was indicated by the calf
kicking at its flanks and dyspnoea with a double expiratory ef-
fort had commenced. Its respiratory rate quickened from 16
per min before injection to 36 per min and its heart rate
similarly from 36 to 68 per min. On an electrocardiograph
taken 2 h after injection, the amplitudes of some QRS com-
plexes and T waves were almost double those before injection
and the polarity of some QRS complexes was reversed. The
dyspnoea persisted and the calf collapsed and died 4 h after
the injection of toxins. No samples were obtained for clinical
pathology. No lesions were seen at necropsy. Both control
calves remained normal.
This study demonstrated that dosing with bryotoxin A
reproduced the effects of eating the plant itself, particularly
the diarrhoea and omasal ulcerations over which there was
some doubt (McKenzie and Dunster 1986, 1987). This con-
firmed that the bufadienolide bryotoxins are responsible for
the toxicity of B. tubiflorum and that the presence of no other
toxin needs to be postulated to account for the syndrome.
Renal dysfunction resulting in the steadily increasing
plasma urea and creatinine concentrations and decreasing
plasma calcium concentrations may have been caused through
a reduced glomerular filtration rate by either autonomically-
mediated renal vasoconstriction, renal vasoconstriction from

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Time after dosing (hours) Figure 2. Changes in the plasma concentrations of urea,
creatinine, calcium and total bilirubin of a calf given bryotoxin
Figure 1. Changes in PR (0) and QT (X) intervals of the elec- A. The first datum point on each curve represents the mean
trocardiograph of a calf given bryotoxin A. The first datum value of 3 daily samples taken before dosing. The horizontal
point on each curve represents the value obtained before dos- lines represent the margins of the normal range of these values
ing. in cattle of similar age.

Australian Veterinary Journal, Vol. 66,No. 11, November, 1989 37s

the direct effects of the cardiac glycoside on vascular smooth
muscle or by poor blood flow from the intoxicated heart.
Renal dysfunction also may have caused the omasal lesions.
Such non-renallesions of uraemia are not described in the pro-
minent texts on veterinary pathology (Jones and Hunt 1983;
Jubb et a1 1985) but have been seen in ruminants with renal
failure (AA Seawright personal communication). The non-
renal lesions recognised in cattle with uraemia are colitis and
oedema of the stomach and proximal intestines (Jubb et a1
1985). Dogs and cats with chronic uraemia develop ulcerative
necrotic stomatitis (Jubb et a1 1985).
The increases in plasma bilirubin and the hepatic changes
detected are more difficult to accept as the results of simple
cardiac dysfunction. Liver damage has not been recorded in
natural cases of Bryophyllum spp poisoning (McKenzie and
Dunster 1986) but one calf dosed with B. daigremontianum
flower heads developed cholangitis (McKenzie et al 1987).
The rapid death of the calf injected with bryotoxins B and C
combined probably was due partly to rapid absorption from
the rumen mediated by the DMSO solvent used. The syn-
drome produced was almost identical with fatal poisoning of
calves given flower heads of the hybrid B. daigremontianum x
B. tubiflorum (McKenzie et all987). These hybrid flowers ap-
parently contained only bryotoxins B and C and the dose given
would have supplied about 660 mg (9.5 mg/kg body weight) of
toxins (McKenzie et a1 1987) - more than twice the dose given
in the present study.
We thank SJK Peucker and AD Ostrofski for help with calf
husbandry, WH Ward for haematology, HL Kramer and staff
for clinical chemistry, G Brown for help with toxin extraction,
K Schultz and G Madill for histological sections and RJ
Capon, Research School of Chemistry, Australian National
University for supplying pure bufadienolides for confirming
the purity of our extracts. This work was partly supported by
the Queensland Department of Primary Industries New In-
itiatives Fund.
References
Anon (1985)-Code of Practice for the Care and Use of Animals for
Experimental Purposes, National Health and Medical Research
Council/CSIRO/Australian Agricultural Council, Australian
Government Publishing Service, Canberra.
Capon RJ, Macleod JK and Oelrichs PB (1985)-J Chem Res (M) 11:
3666
Capon RJ, Macleod JK and Oelrichs PB (1986)-Aust J Chem 39:
1711
Jones TC and Hunt RD 11983b--Veterinarv Patholom. 5th edn. Lea
-_.
and Febiger, Philadelphia ’
Jubb KVF, Kennedy PC and Palmer N (1985)-Pathology of
Domestic Animals. 3rd edn. Academic Press. Orlando, Florida
McKenzie RA and Dunster PJ (1986)--Aust Vet J 63: 222
McKenzie RA and Dunster PJ (1987)-Aust Vet J 64: 21 1
McKenzie RA, Franke FP and Dunster PJ (1987)--Aust Vet 564: 298
(Accepted for publication 14 June 1989)
Urinalysis in captive koalas
Department of Veterinary Pathology, PJ CANFIELD
University of Sydney, DR GEE
New South Wales 2006 DI WIGNEY
One of the more common disease problems facing captive
and free-living koalas is cystitis (Brown et al 1987; Canfield
1987). Cystitis is responsible for frequent micturation and soil-
ing around the opening of the common vestibule (Dickens
1975) and is commonly diagnosed without laboratory testing.
However, laboratory tests are needed to determine ap-
propriate treatment and the possibility of complications for
affected koalas. Biochemical and haematological data in cases
of uncomplicated cystitis and cystitis complicated by renal or
genital disease have been presented by Dickens (1978) and
Obendorf (1983) but these two authors present limited infor-
mation on urinalysis, despite both acknowledging its
usefulness. Hesitation to use urinalysis could be due to the
376
lack of information on urinalysis in the healthy koala. This ar-
ticle attempts to redress that situation by providing data on
urinalysis in captive healthy koalas. In addition, some data are
provided on captive and free-living koalas suffering with un-
complicated and complicated cystitis.
Urinalysis was performed on twenty urine samples collected
over seven visits from 14 healthy captive koalas held in a
wildlife park. One animal had urine collected three times while
four had it collected twice. Eleven of the samples were pro-
cessed for bacteriology. The animals were 19 months to 5
years of age and were fed on eucalyptus leaves with fluid sup-
plementation (a milk compositemade up in about 100-150 mls
of water), Eight were female and six male. In addition,
urinalysis was performed on two adult female captive koalas
with external signs of mild cystitis. Limited results for
urinalysis and bacteriology were obtained also from one 18
months old male and two adult females free-living on the
north coast of New South Wales. All 3 were ultimately
presented for necropsy due to cystitis complicated by renal or
genital disease. Urine from captive koalas was collected early
morning just after feeding of supplementary fluids.
Midstream voided samples were collected in clean disposable
kidney dishes, transferred to sterile urine containers and chill-
ed at 4.C until examined. Catheterisation was attempted on
one visit, but discontinued as koalas with any urine in their
bladder micturated during the catheterisation process.
Urinalysis was performed within three hours of collection.
Gross examination of urine was performed in a graduated test
tube. Specific gravity was determined, using a hand-held
refractometer (American Opticals TS Meter), on urine diluted
with distilled water (one to one or one to two). Turbid urine
was centrifuged prior to this procedure. Chemical analysis was
performed using Labstix, Clinitest, Acetest, Ictotest (all Ames
Co, Miles Laboratories Australia), Whatman pH paper and
the sulphosalicylic acid (SSA) test for protein (Varley 1967).
Sediment was examined according to previously prescribed
methods (Fairburn et al 1983) except that the concentration
ratio was varied (one in two to one in 20) depending on the
amount of urine collected.
For bacteriology, air-dried urine smears were examined with
Burke’s modification of the Gram Stain. Urine was cultured
using a 5 mm platinum loop to inoculate 5% sheep blood agar
plates. The plates were incubated for 24 h and organisms were
identified by colony morphology and Gram Stain. In addition,
routine biochemical tests (Cowan 1975) and motility were
employed for the identification of Enterobacteriaceae.
The amount of urine collected from healthy captive koalas
varied from one to 41 ml. Sex and age differences were not ap-
parent. There was a faint to strong odour, presumably related
to eucalyptus metabolism. The urine appearance varied from
transparent to turbid and was usually light to moderate
yellow, One sample was light amber. Specific gravity varied
from 1.062 to 1.135 with a mean of 1.087. The koala with a
reading of 1.135 was not being given supplementary fluids.
The Labstix gave a consistent pH reading of 5 whereas What-
man’s pH paper (4-6) gave values ranging from 4.5 to 5.5 with
a mode of 5 . Urine samples tested by Labstix were negative for
blood and bilirubin. Protein readings on the Labstix varied
from 0.3 g/l (+) to over 20 g/l (4+) with a mode of one g/l
(2 +). The corresponding SSA tests were all negative for pro-
tein except for a trace on one occasion (corresponding to a 2+
reading on the Labstix). Ketones on the Labstix varied from
0.5 mmol/l (trace) to 16 mmol/l (large) with a mode of 4
mmol/L (moderate). The Acetest for ketones, however, was
consistently negative. Glucose on the Labstix was negative ex-
cept for one trace ( 5 mmol/l). In contrast, the Clinitest gave
values ranging from a 0.25% (trace) to 1.00% (3+) with a
mode of 0.75% (2+).
Variably concentrated sediment generally had uniden-
tifiable debris, scattered squamous and transitional cells, and
erythrocytes and leukocytes at less than one per high powered
field (HPF; 400 x magnification). Amorphous crystals, round
crystals (resembling calcium carbonate) and small elongate
rectangular crystals (Figure 1) were present in 12 samples. The
last described crystals were not identified as they failed to
Australian Veterinary Journal, Vol. 66, No. 11, November, 1989