NEWS AND REPORTS

THE VETERINARY RECORD SEPTEMBER 24, 1994, VOL 135 PAGES 290-291

ANIMAL WELFARE
GOVERNMENT ANNOUNCES NEW ANIMAL EXPORT RULES

NEW controls on live animal exports were announced by Mr William Waldegrave, the agriculture minister, last week.
They are intended to 'improve the protection of farm animals during transport on long journeys' and, for the first time,
include a code of practice for hauliers.

Under the new arrangements, hauliers will have to submit a journey plan for all journeys over 15 hours, giving details of
departure and estimated arrival times, and rest and watering stops at suitable intervals across Europe, not exceeding 15
hours. These plans would then have to be approved by a Government veterinarian.

Mr Waldegrave stated that new legislation would also be introduced to make submission of a false journey plan a criminal
offence. He said that this, together with better arrangements for checking consignments which have been established with
other European Union member states, should improve significantly the chance of successfully prosecuting transporters
who do not carry out their commitments on feeding and watering after crossing the Channel. He said, 'The Government is
seriously concerned at reports that some exporters have not been observing the requirements on feeding, watering and
resting during transit. It has been very frustrating that we have not been able to enforce the law as effectively as we would
have liked. Under my proposals we will be able to do so. The new controls would also give ferry companies the assurances
they need that there is an enforceable regime in place.'

Cool response

However, the ferry companies themselves have been cautious in their responses to the new proposals. Both Stena Ferries
and Brittany Ferries, which have recently stopped carrying live animal consignments, have said that their bans on the
transportation of live animals will stay in place. P&0, which plans to stop carrying animals for slaughter from October 1,
has said that it would review its decision only if enforceable legislation were enacted.

The RSPCA has also voiced doubts as to whether the proposed legislation would actually work and remains 'disappointed'
that the draft does not include a maximum journey time of eight hours for slaughter animals. It said that even existing
weak legislation is 'routinely flouted' and that teams of trained inspectors would be needed to enforce the law properly.

In the past, RSPCA undercover inspectors have followed journeys of up to 60 hours during which animals have not been
given rest, food or water. Animals have been taken to countries other than those stated on official documents. On one
recent journey, for example, a consignment was taken to Portugal instead of the stated destination of the Netherlands.

Mr Peter Davies, director general of the RSPCA, said, 'We welcome the idea of new legislation to improve the welfare of
animals, but the idea of a code of practice simply does not meet our requirements. The only way to ensure the welfare of
animals in transit is to introduce tough European legislation which can be properly enforced.'

The RSPCA will be submitting a formal response on the new proposals to MAFF shortly, and representatives of the
organisation in Paris have called on the French government to persuade them to join Germany, the Netherlands, Denmark
and Belgium in supporting the principle of a maximum journey limit.

Mr Elliot Morley, Labour spokesperson for animal welfare, said last week that the Government should also be
encouraging the export of British carcasses rather than livestock. He said, 'Our lamb is the best in the EU, yet we are
happy for other member states to kill it in their country and then call it their own. If we export carcasses we are not only
stopping the suffering of journeys but also keeping jobs in the finishing of meat at home.'

Farming organisations have also being consulted about the new proposals. Earlier this month, Sir David Naish, president
of the National Farmer's Union, said that 'proper legislation, strictly applied and effectively policed throughout Europe',
was needed. He highlighted the concerns of the livestock industry about the future security of the trade in live animals. He
said that curbing live exports would not be of benefit to animal welfare. 'Continental consumers currently require locally
slaughtered meat. If that demand cannot be satisfied with high quality animals from Britain, then they will source them
from elsewhere -from Russia, Poland and other Eastern European countries where animal welfare is not a priority as it is
here.' The NFU favours the setting up of a new licensing and registration scheme for animal transport operators, both in
the UK and in Europe. Lairages would also be covered by the scheme, with penalties for any operator which fell below
standard.

Negotiations in Europe
Mr Waldegrave agreed that 'tough regulation' was needed throughout Europe. 'Otherwise,' he said, 'we will just penalise
British farmers, whose standards are high, and help some of their rivals who may well have lower standards. That could
have the opposite effect on animal welfare to what we want.' He said that the UK had pressed for tougher Community
sanctions against livestock hauliers who break the rules, and a proposal for licensing hauliers, but added that 'further hard
negotiations' lay ahead.

DOGS
BRITISH STANDARD FOR DOG MUZZLES

The British Standards Institution has published a specification for dog muzzles. Its recommended design is intended to
reduce the effect of serious injuries caused by dog attacks, while taking into account the welfare of the dog.

The Standard has been prepared following consultation with a wide range of organisations, including the British
Veterinary Association, the Metropolitan Police, MAFF, the RSPCA and the Faculty of Veterinary Science at the
University of Liverpool. It will provide manufacturers and other interested parties with a standard design of muzzle.

The BSI recommends that the muzzle should consist of a lattice design cage enclosure which includes a front piece and
adjustable straps to fit the muzzle to the dog's head. It states that box or sock muzzles should not be applied for general
use, and especially not during exercise. It warns that a dog can die if unable to open its mouth to pant or drink, or if vomit
cannot drain from the muzzle.

Each muzzle designed to the Standard will have to be clearly marked with the maximum bodyweight of the dog for which
it is suitable. Manufacturers will also be expected to provide a leaflet giving details of the type of dog for which the
muzzle is intended, whether or not it permits the dog to drink, and information on the fitting and cleaning of the muzzle.
Muzzles designed for short-nosed dogs, such as boxers, should include an adjustable strap from the bridge of the muzzle
to the neck strap.

The Standard states that the muzzle must not prevent the dog from opening its mouth to pant and drink. As a guide as to
how this should be achieved, it says that the vertical depth of the muzzle should be approximately twice the vertical depth
of the dog's muzzle when closed. It adds that the front of the muzzle, when correctly fitted, should not touch or cover the
dog's nostrils, eyes or eyelids.

At least 50 per cent of the surface area of the lattice cage enclosure must consist of air spaces that are unobstructed by any
other components of the muzzle, such as the inner straps. At least 50 per cent of these must be in the lower half of the
muzzle to allow drainage of secretions or vomit.

The Standard indicates that under normal circumstances, the muzzle should be removed when a dog needs to drink, as it
is difficult for it to drink when wearing a muzzle. It adds, however, that dogs which have been fitted with a muzzle in
order to comply with the Dangerous Dogs Act 1991 should be fitted with one that allows them to drink with it on, as,
under the Act, muzzles cannot be removed from these dogs in a public place.

The Standard also gives details on a method for testing the breaking strength of the muzzle, as well as the fitting, use and
care of the muzzles.

 BS 7659; l994 Specification for Dog Muzzles is available from the British Standards Institution, Customer
Services, 3 Carters Lane, Kiln Farm, Milton Keynes MK11 3ET, telephone 0908 221166, fax 0908 260925.

NEWS AND REPORTS

THE VETERINARY RECORD SEPTEMBER 24, 1994, VOL 135 PAGES 292-294

SCOTTISH AGRICULTURAL COLLEGE VETERINARY SERVICES

SUMMER HEAT-WAVE CAUSES PROBLEMS FOR PIGS

The warm weather finally arrived in June but that in turn brought its own problems; temperatures reaching tropical
proportions were recorded inside sow arks according to the Scottish Agricultural College's Veterinary Services' report for
the month. The dry weather was also blamed for dermatitis of the hind digits in bullocks. It is in early summer that young
pheasants are being reared and released and a range of problems was seen in them.
PIGS
GENERALISED AND SYSTEMIC CONDITIONS

A finishing unit buying pigs from two sources suffered severe losses due to a combination of meningitis and polyarthritis
caused by Streptococcus suis type II, sub-acute salt poisoning and colitis. The colitis was associated with Salmonella agoni
and affected the pigs from only one of the suppliers, which was a minimal disease herd. The other pigs came from a herd
known to be infected with S suis.

ALIMENTARY TRACT DISEASE

Loss of condition in pigs from several units was caused by porcine intestinal adenomatosis. Animals from two farms
showed gross lesions in the colon only. Escherichia coli (Abbotstown) was thought to be the cause of scour in 10-day-old
piglets. In another two herds which were investigated the diarrhoea was associated with coccidiosis. Rotavirus was also
present in these pigs. Post mortem examination of two scouring five- to six-week-old weaners revealed a necrotising
colitis in both. The only pathogen isolated was a group B salmonella but the role of this organism was not clear. There
was no evidence of porcine intestinal adenomatosis or spirochaetosis.

Seven finishing pigs in a 600 sow unit died suddenly. Post mortem examination of two pigs revealed lesions of acute
swine dysentery.

RESPIRATORY TRACT DISEASES

Respiratory disease continued to be a problem in many finishing herds with combined infections, for example, enzootic
pneumonia, porcine respiratory and reproductive syndrome, swine influenza, Pasteurella multocida and Actinobacillus
pleuropneumoniae.

MASTITIS

Seven sows died from acute necrotising mastitis and a non-typable E colI was isolated. Many others suffered from a
condition similar to the mastitis-metritis-agalactia syndrome in an outdoor herd. The sows were fed 2 kg/hEad of meal
daily and were in extremely good bodily condition: plenty of grass was available in the paddock. However, temperatures in
some of the arks had reached almost 50 o C.

SKIN DISEASES

The owner of a 400-sow outdoor unit complained of a high prevalence of partial condemnations of the skin which reduced
the weight of each carcase by up to 6 kg. Many of the pigs submitted, which were kept on straw, had a generalised skin
rash. Biopsies taken before the pigs reached the scalding tank revealed epidermitis and dermatitis suggestive of a parasitic
type of infection. The herd in question was a minimal disease herd, free from mange, and the condition seen was thought
to be due to trombiculid mites.

In another case, an owner of a 350-sow indoor herd noted erythematous skin lesions on two of his finishing pigs. Both
showed multiple discrete reddened areas 3 to 5 cm in diameter uver theu backs, flanks and rumps. The lesions took on a
more intense coloration upon excitement and particularly at stunning but disappeared completely upon exsanguination.
Biopsies taken from these lesions showed proliferatiun and dilatation of capillaries in the superficial dermis. In addition
there was a mild subacute to chronic superficial perivascular dermatitis in these areas and in the adjacent normal-looking
skin. The diagnosis considered most probable was vascular hamartomas which could have been congenital, or of
spontaneous later development, with a superimposed chronic hypersensitivity-type dermatitis, possibly parasitic in nature.
Although this was not a major disease problem it caused concern to the abbatoir managers who were initially worried
about the health of the pigs. However, once the blemishes had disappeared the inspectors were unable to distinguish the
affected pigs from other normal animals.

CATTLE
GENERALISED AND SYSTEMIC CONDITIONS

The Salmonella typhimurium phage type 104 epidemic appeared to be slowing down this month. Only 14 incidents were
reported from four centres compared with 25 incidents in May. Edinburgh reported the most interesting outbreak, which
involved a group of 30 in-calf Holstein-Friesian heifers imported from Holland. Two days after arrival on the farm one
animal died after exhibiting pyrexia, enteritis and oral ulceration. S typhimurium phage type 104 and bovine viral
diarrhoea (BVD) virus were isolated from the carcase. 'The salmonella organism was also isolated from three other
heifers, two of which were ill. Fifteen of the 29 survivors seroconverted to BVD virus.

In one other outbreak of acute BVD infection one animal died and two of the six animals tested seroconverted to BVD
virus. They came from a group of 22 Friesian heifers aged six to nine months which had exhibited salivation, scour and
oral ulcers.

TOXIC CONDITIONS

Five of the eight centres reported incidents of lead poisoning in cattle. In three of the seven cases no source of lead was
identified, but in one of them suspicion was cast on urea fertiliser. The affected cattle had grazed a field treated with the
fertiliser a few days previously. The farmer reported seeing black necks in what should have been an homogeneous white
product. Analysis of a small sample of the urea from an unopened sack revealed a lead concentration of 7 parts per
million, a level not thought to be toxic and well below the concentration found in some soils, but there should be no lead
in urea. Two Friesian heifers from a group of 15 died of yew poisoning after children carried branches from a yew tree
into the field.

ALIMENTARY TRACT DISORDERS

June was a quiet month for enteritis submissions, but four centres reported outbreaks of coccidiosis. Two new incidents of
necrotising enteritis of suckled calves were diagnosed.

RESPIRATORY TRACT DISORDERS

June is not usually a month associated with respiratory disease, but four centres reported outbreaks and implicated
respiratory syncytial virus on five occasions. Infectious bovine rhinotracheitis was also diagnosed and Haemophilus
somnus was isolated from the pneumonic lung of a 10-week-old housed dairy calf, the third such animal to die.

NERVOUS SYSTEM DISORDERS

A dairy head experienced an outbreak of central nervous disease in adult cows. The first three were slaughtered as suspect
BSE cases and two of these were diagnosed as suffering from cerebrocortical necrosis. The result of the third examination
is awaited. A fourth animal killed and auotpsied on the farm had a hepatic encephalopathy. One individual was treated
with thiamine and recovered. No explanation for the outbreak of cerebrocortical necrosis was found.

Focal symmetrical encephalomalacia was identified as the cause of death in two 10-week-old suckled calves in the same
herd. No signs of illness had been observed. In a case from another herd clinical signs were observed, but initially
attributed to complications following disbudding two weeks previously.

SKIN DISEASES

An outbreak of dermatitis affecting the skin immediately above the coronary band on the lateral aspects of the hind digits
was observed in young grazing bulls in the Dumfries area. Microbiological investigation identified Staphylococcus aureus
from all sampled lesions and Bacteroides melaninogenicus was isolated from two. Examination by electron microscopy
failed to show any virus particles. On histological examination of biopsy material, lesions resembled those seen in
interdigital dermatitis. Only bulls were affected, ground conditions were dry and there appeared to be a good response to
antibiotic therapy. Very similar outbreaks have been observed previously in dry summer conditions at both Dumfries and
St Boswells. The aetiology remains obscure.

Ayr identified a case of photosensitisation attributed to congenital erythropoietic protoporphyria in a Limousin heifer. The
serum protoporphyrin concentration was above 400,000 nmol/litre in the affected individual compared with 1500 and
2300 nmol/litre in two unaffected calves from the group. This condition has been reported from other countries, but had
apparently not been recorded in the UK.

REPRODUCTIVE TRACT CONDITIONS

Mycotic infection (four reports), BvD (two reports) and Bacillus licheniformis (one report) were implicated in bovine
abortions.

SHEEP
Nutritional and metabolic disorders

Hypocupraemia associated with ill-thrift was confirmed in three flocks by Thurso while Dumfries found low vitamin B12
levels in a single flock.

The explosive outbreaks of nematodiriasis seen in May settled down a little in June but deaths in lambs associated with
Nematodirus battus were recorded in five flocks by Ayr and four by Dumfries. Nephrosis was sometimes a complication.
In many flocks other trichostrongyle worms also featured to a significant extent. One lamb had a worm egg count of
13,500 N battus and 44,000 trichostrongyle eggs per gram while coccidiosis caused the death of a lamb at Thurso. A 14-
week-old pet lamb that had been scouring and was in poor condition prior to death was shown by Dumfries to have a
heavy burden of Strongyloides papillosus.

Alimentary tract disorders

At Aberdeen a lactating ewe died from a strangulated inguinal hemia and redgut was confirmed in a sucking lamb. On a
farm in the Dumfries area there had been five recent deaths in blackface gimmers. Two were submitted and both showed
torsions about the root of the mesentery.

Respiratory tract diseases

Relatively minor outbreaks of pneumonic pasteurellosis were reported by most centres. Chronic pneumonia in a group of
ewes was suspected by the veterinary practitioner as being sheep pulmonary adenomatosis (SPA) but laboratory
investigations showed Actinomyces pyogenes and no histological evidence of SPA. At St Boswells a gimmer which
exhibited terminal pasteurella septicaemia had underlying lesions suggestive of parainfluenzavirus 3 pneumonia.

Musculoskeletal conditions

An adult ewe with chronic arthritis in several joints yielded a mixture of Streptococcus agalactiae and S dysgalactiae from
joint fluid and the latter organism from the spleen. Also at Aberdeen, S dysgalactiae was associated with septic arthritis in
five of 70 lambs.

Nervous system disorders

Listeriosis was diagnosed at Perth in a shearling tup showing evidence of a nervous disorder before death and in four
shearlings from a group of 32 at St Boswells where a suppurative meningoencephalitis was confirmed. Two cases of
cerebrocortical necrosis were diagnosed at Edinburgh and one at St Boswells, one case in a ewe and two in lambs.

Skin diseases

Sheep scab was confirmed at St Boswells, Perth and Dumfries in single flocks.

A cautionary tale

A farm 100 miles from Ayr lost 300 lambs in the first three months of life; the SAC report notes that the farmer was
unwilling to drive the distance to deliver carcases. Because the lambs showed nervous signs, two brains were finally sent
in for histological examination. No abnormalities were seen. The report adds that a thorough post mortem examination
might well have identified the problem.

Goats

Enterotoxaemia was confirmed in two adult goats at Aberdeen. An Anglo-Nubian goat had mastitis in both quarters and
Streptococcus uberis was isolated.

Birds

Poultry
Inclusion body hepatitis was diagnosed in 29-day-old broilers in which there was rising mortality. At post mortem
examination a consistent findmg was the presence of an enlarged pale liver with reticulate pattern and inspissated bile in
the gall bladder. Cultures from the liver and spleen demonstrated Clostridium perfringens. Many eosinophilic and
basophilic intranuclear inclusions were visible in the hepatocytes on histopathological examination of the liver.

Broiler ascites syndrome was reported by the Perth, Ayr and Thurso centres. Severe caecal coccidiosis resulted in deaths in
eight week-old broiler breeder replacements, and losses in broiler breeder cockerels were caused by an acute outbreak of
necrotic enteritis.

A drop in egg numbers, the production of eggs with pale shells and increased mortality occurred on a multiage battery
cage laying site. Airsacculitis, pericarditis, perihepatitis and egg yolk peritonitis was found on post mortem examination
of affected birds. There was serological evidence of exposure to infectious bronchitis virus 793B. Acute Marek's disease
was also present in some birds from this site.

Aspergillosis was diagnosed in turkeys aged eight weeks which were run on straw litter that appeared mouldy. Multiple
white granulomata and plaques were present on the lungs and air sacs, and Aspergillus species was isolated on culture.

Gamebirds

Salmonella binza was isolated from pheasants by the Ayr, Dumfries and Perth centres.

Gamebird submissions were dominated by rotavirus infections in young pheasants, often with accompanying E coli.
Aspergillosis accounted for substantial losses in young chicks from a rearing field where the disease had been diagnosed
in previous years. Kidney failure, starve outs and yolk sac infections were also found in young birds. As the pheasants
grew older, losses associated with the use of plastic bits in young birds were reported. The resulting feeding problems
caused starvation and secondary colispeticaemia. The farm was visited and the appropriate advice given, and the problem
was resolved.

Trichomoniasis was found affecting one batch of pheasants aged three to four weeks, and hexamitiasis in another batch of
birds of a similar age.

Pigeons

Paramyxovirus infection was confirmed by virus isolation on a young pigeon from an Edinburgh loft in which
unvaccinated birds were showing signs of a central nervous disorder.

Trichomoniasis was diagnosed in pigeons from a number of lofts wiLh signs of ill thrift, failure to perform, or loose
faeces.

A combination of candidiasis and hexamitiasis was found in a pigeon from a loft in Ayr, where young birds were loosing
weight and had diarrhoea. Chlamydiosis resulted in similar signs in young birds from a different loft; at post mortem
examination airsacculitis and perihepatitis were present, and chlamydia was demonstrated by the polymerase chain
reaction.

Cage and aviary birds

Four of the commonest problems seen in cage and aviary birds are megabacteriosis, trichomoniasis, chlamydiosis and
pulmonary aspergillosis. All were seen in June, the first two affecting budgerigars and the last two affecting parrots.

Wildbirds

Seven of a brood of eight cygnets in Dumfries died before they were a month old. One cygnet was found to have large
numbers of tapeworms in the intestines.

Miscellaneous mammals
Dogs and Cats

Ayr, Edinburgh and Perth all reported the isolation of Campylobacter species from faecal samples from diarrhoeic dogs.
Ayr commented that the human contacts with an 11-month-old samoyed were also affected by diarrhoea. Dumfries
isolated Campylobacter jejuni and Clostridium di@icile from the faeces of a nine-year-old cat suffering from chronic
diarrhoea.
Ringworm was recorded by cats in Ayr and in a seven-year-old boxer puppy by Dumfries. The latter centre confirmed
sarcoptic mange in a four-year-old lurcher. Among the more interesting cases investigated was infectious canine hepatitis
in one of two unvaccinated, three-year-old Yorkshire terrier dogs. The remaining dog was never ill.

Horses

ed in a diagnosis of fascioliasis in a 13-year-old mare at Dumfries while Edinburgh found a small intestinal volvulus in a
six-week-old Clydesdale foal.

Deer

Yersinia pseudotuberculosis was isolated by St Boswells from a mesenteric lymph node abscess found in a wild roe deer.
The one-year-old animal had been found dead and was autopsied by a practitioner.

Bats

A faecal sample was submitted to Dumfries from a household with 2000 bats because the owner was concerned to know
whether the bats were carrying bacteria pathogenic for humans. The sample was examined for rotavirus and
cryptosporidia but was negative. On culture, Bacillus species and Staphylococcus aureus were the only bacteria isolated: is
this normal bat intestinal flora?

Fish

'Trout fry anaemia' (Cytophaga species septicaemia) was the cause of a 1 per cent per day mortality among juvenile
rainbow trout. There were characteristic histological lesions of splenic necrosis and congestion, and numerous typical
small long Gram-negative bacteria were present on stained spleen smears. This infection appears to be becom
ing more widespread.

Frenzied feeding activity appeared to be predisposing factor in an outbreak of enteric redmouth (Yersinia ruckeri
infection) among older rainbow trout. This infection was also identified as a cause of mortality among rainbow trout
reared in sea-cages.

A series of sudden ovemight mortalities occurred among salmon fry reared in tanks on an artificial photoperiod regime. It
seemed likely that the abrupt change back to light caused a fright reflex, with crowding and asphyxiation. The problem
did not recur once continuous lighting was introduced.

The change in water chemistry when spates followed a long dry period may have predisposed salmon parr to a serious
bacterial gill disease problem. Those introduced to the farm one month previous1y from a similar soft-water area
experienced little problem, while heavy mortality occurred among others which came from a hard-water area.

A similar problem of severe gill hyperplasia with myxobacterial colonisations occurred elsewhere when salmon parr and
smolts were moved from tanks to ponds, but with furunculosis subsequently appearing among the stock.

Early summer traditionally heralds problems among ornamental pond fish, and this year was no exception. The Perth
centre reported heavy mixed ectoparasite infestation including the protozoans Costia, Ichthyophthirius (white spot) and
Chilodonella along with Dactylogyrus and Gyrodactylus gill and skin flukes among a group of koi carp, with Trichodina
protozoa and Gyrodactylus parasitising goldfish at another location.

LETTERS
THE VETERINARY RECORD SEPTEMBER 24, 1994, VOL 135 PAGE 316

URAEMIA IN CASES OF CRYPTOSPORIDIOSIS

V.R.Simpson, MAFF Veterinary Investigation Unit, Polwhele, Truro, Cornwall TR4 9AD

SIR, in their recent short communication Molina and others ( VR, July 16, p67), record elevated serum urea levels in goat
kids suffering from cryptosporidiosis. In discussing this they state that the increase could have been 'due to increased
protein catabolism, which has also been observed in red deer (Simpson 1992)'. There would appear to be some
misunderstanding here for, although I accept that increased protein catabolism is a possible explanation for the raised
urea levels, it was not one which I proposed in my article. The explanation that I put forward was that the uraemia arose
as a result of renal insufficiency, possibly as a result of electrolyte loss into the intestine.

During that investigation into red deer (Simpson 1992) it was found that there was no significant difference between urea
levels in serum, cerebrospinal fluid and aqueous humour. The aqueous humour urea levels in the red deer calves ranged
from 50 to 147 mmol/litre (301 to 885 mg/100 ml). In subsequent years I have examined aqueous humour urea levels in
calves and lambs dying from enteritis during the first three weeks of life. This has shown that high urea levels commonly
occur in animals suffering from Cryptosporidium species infection, but are seen much less frequently in association with
other enteric pathogens, such as rotavirus, Escherichia coli and Salmonella species. This is illustrated by an analysis of 29
consecutive cases during 1992/1993. In 12 cases (nine calves, three lambs) where cryptosporidia were not demonstrated
the mean urea level was only 10.3 mmol/litre (sd + 3.5), whereas in 17 cases ( 12 calves, five lambs) where cryptosporidia
were demonstrated the mean aqueous humour urea level was 34.5 mmol/litre (sd +- 8.4). Although these values are
generally lower than those reported in the red deer calves, they are considerably higher than the values reported by Molina
and others in infected goat kids mean value 7.98 mmol/litre, sd + 3.55).

It is also interesting to note that values in lambs were consistently higher than in calves, and ranged from 32 to 68
mmol/litre. In one such case a farmer reported nine- to 10-day-old lambs suddenly going 'floppy' and dying. There was no
significant diarrhoea, but the lambs showed severe cryptosporidiosis and the aqueous humour urea levels were 60 and 66
mmol/litre, respectively. It would appear, that the clinical signs were principally due to the severe uraemia and the
condition in these lambs therefore closely resembled that seen in the red deer calves.

I would be interested to hear whether similar findings have been seen elsewhere.

SHORT COMMUNICATIONS
THE VETERINARY RECORD SEPTEMBER 24, 1994 VOL 135 PAGE 308-309

ISOLATION AND IDENTIFICATION OF MYCOPLASMA MYCOIDES

SUBSPECIES MYCOIDES SC FROM BULL SEMEN AND SHEATH
WASHINGS IN PORTUGAL
M Rosario Goncalves, Department of Bacteriology, Laboratorio Nacional de Veterinaria, Estrada de Benfica 701, 1 500
Lisboa, Portugal

MYCOPLASMAS are common hosts of mucosal surfaces and are frequently isolated from the urogenital system, although
the presence of these agents and the diseases associated with infertility or other disturbances has not yet been clarified.

Mycoplasma mycoides subspecies mycoides is divided in two closely related biotypes: LC and SC, the latter being the
aetiological agent of contagious bovine pleuropneumonia (CBPP).

The purpose of this communication is to report the isolation of Mycoplasma mycoides subspecies mycoides SC, from
semen and preputial washings of two 11-month-old bulls and from the preputial washing of another bull not associated
with subclinical or clinical CBPP. The bulls in this study were the result of imported frozen embryos implanted into
Portuguese cows and were being considered for entry to a breeding centre.

The two semen samples and the three saline sheath washings were received for bacteriological examination within 24
hours. All the samples were cultured for Mycoplasmas using media and techniques described previously (Goncalves
1990). Briefly 2 ml of medium B (Erno and Stipkovits 1973) and 2 ml of medium Ball (Ball and others 1987) in tubes
were incubated with 0.2 ml of the semen samples and the inoculated media were diluted 1:10, 1:100 and 1:1000 and
incubated at 37 C. Each semen sample was centrifuged at 3500 g for 30 minutes. The pellets were cultured by streaking
on Ball agar medium and the plates were incubated in the presence of 6 per cent CO2. Each preputial washing sample
was centrifuged at 600 g for five minutes. The supernatant was filtered through 450 nm Millipore filters and plated on B
and Hayflick media and broth; the pellet was cultured as previously described for semen. Biochemical tests showed that
mycoplasmas fermented glucose, reduced tetrazolium salts, were negative for arginine, had variable phosphatase activity
and did not digest serum or casein. The serological identification of the isolated strains was determined by growth
inhibition (GI) tests. They were positive with rabbit hyperimmune serum to M mycoides subspecies mycoides sc (reference
strain B 17) and negative with bovine serum of a natural case of CBPP (CF titre 1:320), with bovine serum (CF titre
1:2560) inoculated with M mycoides subspecies mycoides SC (field strain PB 90) and with rabbit hyperimmune serum to
M capricolum (B 304 strain) isolated from a bovine.

The protein profiles of M mycoides subspecies mycoides SC (field strain PB 90), other mycoplasmas of M mycoides
cluster and those from bovine genitalia were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis
(Laemmli 1970) in 7 to 22.5 per cent linear gradient gel according to Ferreira and Duarte (1990). The protein profiles of
all of these strains were identical.

The genomic analysis of these mycoplasmas, M mycoides subspecies mycoides SC reference strain B 17, M mycoides
subspecies mycoides LC reference strain Y-Goat, M mycoides subspecies mycoides SC (field strain PB 90), Mycoplasma
PG 50 and California Kid strain using HindIII digestion was also carried out according to the methodology of Razin and
others (1983) with modifications described by Machado and others (1992). Basically, after treatment with phenol the
samples were purified twice using chloroform:isoamylic alcohol (24:1). After precipitation with cold ethanol the DNA was
double treated with saturated ether in water, dried under vacuum and dialysed against 10 mM Tris pH 7.5 containing 1
mM ethylenediamme tetra-acetic acid. In spite of the production of many bands, identical cleavage patterns were
observed, demonstrating genetic homogeneity between B 17, PB 90 and the author DNA strains. These patterns were
unique and different for Y-goat and PG 59.

Samples of blood, semen, preputial washings and urine were collected every 15 days for one year from all the bulls.
Bacteriological culture for the presence of the agent and a CF test for the presence of specific antibodies proved to be
negative throughout this period. During this time and to date, the bulls have remained healthy. Although the pathogenic
role of M mycoides subspecies mycoides sc in the bovine respiratory system is well known, these animals showed no
adverse respiratory tract symptoms or deleterious effects to the male genital tract. The source of these mycoplasmas is
unknown.

Although a reference has been made to the presence of M.mycoplasma mycoides in semen ofruminants (Hare 1985), it
was not referred to as the subspecies. Therefore, the author assumes that this communication is the first report of the
isolation of M mycoides subspecies mycoides SC from bovine semen and preputial washings.

The epidemiological significance of M mycoides infections in ruminants should be re-examined as well as the pathogenic
aspects of the CBPP agent.

------------------------------
ABSTRACTS
THE VETERINARY RECORD SEPTEMBER 24, 1994, VOL 135 PAGE 309

CRYSTALS IN EQUINE ARTICULAR CARTILAGE

HAYES,A.,SWAN,A.J.&DIEPPE,P.A.(1994) Research in Veterinary Science 57, 106

EIGHTY-NINE fetlock joints selected at random from horses slaughtered at a local abattoir were dissected and the
cartilage covering the distal end of the cannon bone was examined for its general condition and for evidence of crystal
deposits. Thirty-six of the joints appeared normal, but the others had evidence of osteoarthritis, or scratch marks parallel
to the direction of motion within the joint. Ten of the joints contained crystal deposits visible by eye, and an examination
by electron microscopy and X-ray diffraction showed that these crystals were octacalcium pyrophosphate and calcium
pyrophosphate dihydrate. It is suggested that the deposition of crystals may be a mediator of arthritis in horses and may
also cause damage directly.

ABSTRACT
THE VETERINARY RECORD SEPTEMBER 24, 1994, VOL 135 PAGE 309

FEMORAL NECK ANGLE AND HIP DISPLASIA IN DOGS

MASDEN,J.S.& SVALASTOGA,E.(1994)Acta Veterinaria Scandinavica 35, 115

THE mean angle of the femoral neck was measured radiographically in 41 medium to large dogs examined for hip
dysplasia. A steeper than average angle of femoral neck inclination was found to be associated with hip dysplasia and
laxity of the coxofemoraljoint. It is suggested that the bioomechanical changes which result from this abnormally steep
angle may be a factor in the pathogenesis of hip dsyplasia and secondary osteoarthritis.

PAPERS AND ARTICLES

THE VETERINARY RECORD SEPTEMBER 24, 1994 VOL135 PAGES 296-302

EPIDEMIOLOGICAL OBSERVATIONS ON SPONGIFORM

ENCEPHALOPATHIES IN CAPTIVE WILD ANIMALS IN THE BRITISH
ISLES.
J.K.KIRKWOOD;A.A.CUNNINGHAM
Since 1986, scrapie-like spongiform encephalopathy has been diagnosed in 19 captive wild animals of eight species at or
from eight zoological collections in the British Isles. The affected animals have comprised members of the family
Bovidae: one nyala (Tragelaphus angasl@), four eland (Taurotragus olyx), and six greater kudu (Tragelaphus
strepsiceros), one gemsbok (Olyx gazella), one Arabian oryx (Oryx leucoryx), and one scimitar-horned oryx (Oryx
dammah), and members of the family Felidae: four cheetah (Acinonyx Jubatus) and one puma (Felis concolor). In
addition, three cases of a spongiform encephalopathy of unknown aetiology have been reported in ostriches (Struthio
camellus) from two zoos in north west Germany. Three features suggest that some of these cases may have been caused by
the agent of bovine spongiform encephalopathy (BSE). First, they have been temporally and geographically coincident
with the BSE epidemic.

Secondly, in all the ungulates for which details are available, it is possible that either the affected animal itself, or the herd
into which it was born or moved, had been exposed to proprietary feeds containing ruminant-derived protein or other
potentially contaminated mr@terial, and all the carnivores had been fed parts of cattle carcases judged unflt for human
consumption. Thirdly, the pathological results of inoculating mice with a homogenate of fixed brain tissue from the nyala
and from one greater kudu were similar to the results of inoculating mice with BSE brain tissue.

BEFORE 1980, naturally occurring scrapie-like subacute spongiform encephalopathies were known in six species. These
diseases included scrapie in domestic sheep and goats (Dickinson 1976), kuru and other spongiform encephalopathies in
man (Prusiner and Hadlow 1979), transmissible mink encephalopathy in mink (Mustela vison) (Hartsough and Burger
1965), and chronic wasting disease in mule deer (Odocoileus hemionus) (Williams and Young 1980) and Rocky Mountain
elk (Cervus elaphus) (Williams and Young 1982). Since 1980 spongiform encephalopathies have been reported in a
variety of other species, especially species of wild animals maintained in zoos and wildlife parks in Great Britain (Bradley
and Matthews 1992, Wells and McGill 1992).

This paper gives information on all the recent cases of spongiform encephalopathy in captive wild animals of which the
authors are aware. They comprise 19 cases in eight species at or from eight zoological collections in the British Isles. A
similar condition have also been reported in ostriches in Germany (Schoon and others 1991). The paper reviews some of
the epidemiological and other features of the cases, accounts of some of which have been published previously, and
includes information on some which have not been described in the literature. The pathological findings have been
presented in many of the original reports and in some reviews (Wells and McGill 1992) and they are not discussed here.
The paper concludes with a consideration of the control of the disease in wildlife collections.

Information has been collected on the epidemiological aspects of 19 cases of spongiform encephalopathy in captive wild
animals in the British Isles and three from Germany, both from the literature and from discussions with veterinarians and
curators involved with the cases. The data collected included dates of birth and death, history of feeding practices, and
details of any movements between collections and contact with animals of the same or other species. Brief details of the
cases are presented in Table 1. In all these cases, the condition had been diagnosed on the basis of the histopathology and,
in some cases, by the detection of scrapie-associated fibrils (SAF) and/or the disease-specific isoform of the glycoprotein
PrP (Wells and McGill 1992), and by experimental transmission of the condition to mice (Table 2) (Jeffrey and others

THE CASES
NYALA (TRAGELAPHUS ANGASI)

The diagnosis of the scrapie-like spongiform encephalopathy in the nyala (Tables 1 and 2) pre-dated the diagnosis of the
first cases of bovine spongiform encephalopathy (BSE) in cattle (Jeffrey and Wells 1988). Although the clinical and
pathological findings in this case have been described previously (Jeffrey and Wells 1988), little infolmation relevant to
the epidemiology of the disease has hitherto been available. Notes on the clinical signs are listed in Table 3.

TABLE 1: Cases of scrapie-like spongiform encephalopathy in captive wild animals in Britain

Date of Date of Age
Species Animal birth death (months)Sex
Nyala 4. 9.83 18.6.86 33 F
Gemsbok 18. 6.83 8.6.87 48 F
Eland 1 6. 4.87 20.12.89 32 M
Eland Molly 14. 1.89 9. 4.91 27 F
Eland Neddy 10. 1.89 25. 5.9128 M
Eland Electra 12. 1.90 23. 1.9224 F
Arabian oryx 12. 1.86 24. 3.8938 F
Greater kudu Linda 11. 2.87 18. 8.8930 F
Greater kudu Karla 19. 4.89 13.11.90 19 F
Greater kudu Kaz 14. 5.88 6. 6.91 37 M
Greater kudu Bambi 29.10.88 24.10.91 36* M
Greater kudu 346/90 26. 8.90 26. 2.9218* M
Greater kudu 324/90 5. 8.89 22.11.92 39 F
Scimitar-horned oryx 12. 7.90 29. 1.9330 F
Puma 8. 3.86 16. 5.9162 F
Cheetah 1 16. 6.86 7. 1.92 55 M
Cheetah Duke 3. 9.84 27.10.92 96 M
Cheetah Saki 23. 2.86 4. 5.93 86 F
Cheetah Michelle 14. 5.86 22.12.93 91 F

* These animals showed no clinical signs when they were euthanased

The animal was handreared on cow or goat milk and weaned on to a diet of which part was a proprietary concentrate
pellet. This pellet was not thought to contain ruminant-derived protein either before March 1986 or after March 1987. If
this was so, the nyala was exposed to this material only during the three months preceding her death (Wilesmith and
others 1988) and it seems very unlikely that the incubation period could have been this short. The diet may have been
unwittingly contaminated before March 1986, or the animal may have acquired the disease from another source. No other
source is, however, apparent. The nyala herd which numbered 16 animals at the time of this female's birth, was not in
direct contact with any other ung@lates. Sheep were kept at the premises where the animal was handreared but there had
been no cases of scrapie among them. The results of bioassay of brain material from the nyala suggested that the agent
was like that of BSE (see below).

Although both of this animal's parents are now dead, the dam had eight further calves between 1985 and 1991, and no
cases of spongiform encephalopathy have been detected among them. The affected nyala was 33 months old when she
died and had not bred.

TABLE 2: Features on which the diagnosis of spongiform encephalopathy was based in captive wild animals.

Species Animal Histopathology SAF PrP Transmission

Nyala + +
Gemsbok + +
Eland 1 +
Eland Molly +
Eland Neddy +
Eland Electra + +
Arabian oryx +
Greater kudu Linda + +
Greater kudu Karla + + +
Greater kudu Kas + +
Greater kudu Bambi + + +
Greater kudu 346/90 + + +
Greater kudu 324/90 + +
Scimitar-horned oryx + +
Puma + +
Cheetah 1 + +
Cheetah Duke + +
Cheetah Saki +
Cheetah Michelle + +
Ostrich 1, 2&3 +

SAF Scrapie-associated fibrils

In this case, and in the case of the kudu Linda (see below), experimental transmission to mice was attempted by the
inoculation of homogenates of fixed brain tissue, and in both cases the transmission was successful (H. Fraser, personal
communication, Jeffrey and others 1992). The incubation periods observed after the intracerebral and intraperitoneal
inoculations into six strains of mice (Fraser and others 1992) are shown in Table 4. The pattern of variation in the
incubation periods between the strains of mice was the same for both the nyala and kudu (RIII <VM Sinc S7 <C57BL
<VM <IM <C57BL x VMF cross) (H. Fraser and I. McConnell, personal communication, Bruce and others 1994), and
this ranking is the same as has been observed in the transmissions of BSE to mice (Fraser and others 1992, Bruce and
others 1994).

Gemsbok (Oryx gazella)

The gemsbok was a four-year-old female kept at the same premises as the nyala. It was fed the same diet as the nyala and
the diet was thought to contain ruminant-derived protein only from March 1986 to March 1987 - the period three to 15
months before the animal died (Wilesmith and others 1988).

The animal was in good bodily condition at the time of her death, but had had episodes when she suddenly collapsed for a
few minutes, followed by periods of relatively normal behaviour (Table 3).

There were 13 animals in the gemsbok herd at the time of this animal's death. The herd was isolated from other animals
but in contact with some Congo buffalo (Synceros caffer). There had been no contact with the nyala. No further cases of
spongiform encephalopathy have been observed in any animals on the premises.

The affected gemsbok had given birth to one calf, a male born on January 6, 1987, five months before she died. The calf
died on the day it was born. The dam of the affected gemsbok gave birth to a further six calves between 1984 and 1991
and was euthanased as a result of old age.

Arabian oryx (Oryx leucoryx)

There has been one case in an Arabian oryx (Tables 1, 2 and 3), a female born in Zurich in Januar,v 1986 and imported to
Regent's Park in October that year. The herd at Regent's Park was fed a proprietary cattle feed that was quite likely to have
contained ruminant-derived protein and it was considered by Kirkwood and others (1990) that the animal may have
acquired the agent from the feed. Assuming that she acquired the infection after arriving in Britain, the incubation period
was at most 29 months. The animal had one calf, a male born on October 6, 1988, which was exported to Saudi Arabia in
July 1989 but became acutely ill and died on August 18, 1989 after an excessive intake of concentrate (Kirkwood and
others 1990).

TABLE 3: Clinical signs observed in cases of spongiform encephalopathy in zoo animals

Animal Clinical signs Duration

Nyala Hindlimb ataxia, abnorrnal head posture, 21
persistent nibbling of tail base and rump leading
to mutilation and ulceration, frequent micturition

Gemsbok Sudden onset, frequent episodic collapsing, 7

good body condition

Eland 1 High-stepping of hindlimbs, fine muscle tremors 8

of head and neck, loss of weight. Later, circling,
head-pressing, dullness, drooling and clear nasal
discharge

Eland 2, 3 and 4 Weight loss, drooling, standing apart from others, 14-21
slight twitching of flank and intercostal muscles.
Ataxia not a prominent feature, but one showed
slight high-stepping

Arabian oryx Weight loss, muscle tremors and later ataxia 22

and dullness

Kudu Linda Ataxia, head tilt, drooling, excessive lip-licking 3

and nose-twitching, some weight loss

Kudu Karla Ataxia, forelimb crossing, hindlimb hypermetria, 1

head tilt, depression

Kudu Kaz Similar to Linda 1

Kudu Bambi No clinical signs when euthanased for 0

management reasons

Kudu 346/9o No clinical signs when euthanased for 0

management reasons
Kudu 324/90 Initially intermittent head-tilt, mild head tremor 56
excessive lip movements, hindlimb muscle tremors and
hunched posture. Later, decreased abnormal head and
ear carriage, hypermetria possible hyperaesthesia

Scimitar-horned oryx Nasal discharge, cough, weight loss. 18

Later, collapsed

Puma Ataxia, difficulty maintaining balance, fine whole 6

body tremor, 'looking upwards and around in an unusual way'

Cheetah 1 Ataxia, apparent disorientation, falling and 2

locomotory weakness

Cheetah Duke Ataxia, hyperaesthesia. Later, weight loss 31

Cheetah Saki Ataxia ca 42

Cheetah Michelle Proggressive ataxia, tremors, muscle spasms ca 56

and falling

Ostriches Ataxia, disturbance of balance, 'discoordinated 'Protracted'

feeding behaviour'

Greater kudu (Tragelaphus strepsiceros)

The incidence of spongiform encephalopathy in the small herd of greater kudu maintained by the Zoological Society of
London has been described in detail (Kirkwood and others 1990, 1992, 1993, 1994, Cunningham and others 1993). The
temporal and familial relationships of the six confirmed cases are shown in Fig 1. The disease was first confirmed in a
female (Linda) that died (August 18, 1989, and the diagnosis was confirmed by the transmission of the disease to mice
(Table 2). The ranking of the incubation periods in six strains of mice after their experimental inoculation with
homogenised brain was the same as that found for the nyala (see above and Table 4) and the same as that found in the
transmission of BSE (H. Fraser and I. McConnell, personal communication, Bruce and others 1994).

Since then five further cases have been diagnosed post mortem (Tables I and 2). The number of cases in this small herd
suggested that the animals were highly susceptible -the disease has been diagnosed in five of eight animals born at
Regent's Park since 1987 (Kirkwood and others 1993). Furthermore, although the initial case may have acquired the
infection from feed containing ruminant-derived protein the pattern of incidence suggested that the disease may
subsequently have spread by transmission between animals (Kirkwood and others 1992, 1993, 1994, Cunningham and
others 1993).

The diet fed to the kudu at the Zoological Society of London did not contain rurninant-derived protein after February
1987 although proprietary pellets that may have contained the material were present on the premises. It is very unlikely
that the kudu would inadvertently have been given these pellets and, furthermore, the dates of birth of three of the affected
animals born at Regent's Park were three, nine and 25 months after the ban on the inclusion of ruminant-derived protein
in ruminant feeds (Order 1988) (Table 1). Feed storage practices were such that no feed would have been stored for longer
than a few weeks. In the absence of other potential sources of the disease, it was hypothesised that it may have been
transmitted between animals.

TABLE 4: Mean + se incubation periods observed after the experimental inoculation of six strains of mice with brain
homogenates from a nyala and a greater kudu in which scrapie-like spongiform encephalopathy had been diagnosed on
the basis of histopathological signs (H. Fraser and 1. McConnell, personal communication)

Incubation period (days)

Mouse strain Nyala Kudu
Rlll 378 + 8 339 + 5
VM Sinc s7 459 + 14 410 + 8
C57BL 528 + 11455 + 9
VM 548 + 16 536 + 10
IM 614 + 11560 + 12
CS7BL x VM 772 + 3 751 + 24

The method was that described by Fraser and others (1992)

The most recent case was in a kudu (324/90) born 13 months after July 1988 (Tables 1, 2 and 3) at another zoo in
England where the feeds had not contained ruminant-derived protein since March 1987; she was moved to Regent's Park
on August 14, 1990, 27 months before she died (Kirkwood and others 1994). Since the incubation period can be as short
as 19 months in kudu (see details on Karla in Table 1) and there had been no cases in kudu at the zoo where the animal
was born, nor the likelihood that she might have been fed ruminant-derived protein. It was considered that this animal
might have acquired the disease by transmission from other affected animals after her arrival at Regent's Park (Kirkwood
and others 1994).

Eland (Taurotragus oryx)

Four confirmed cases of spongiform encephalopathy have occurred in eland (Tables 1, 2 and 3) (Bradley and Matthews
1992), but only one of them has been described in any detail (Fleetwood and Furley 1990). It was a 32-month-old male
eland which had been fed on a proprietary ungulate ration which contained ruminant-derived protein from calfhood to
June 1988, the authors suggested that this might have exposed the animal to the causal agent of BSE or scrapie.

???????????????????????????????????

TABLE 5: Numbers of species and individual wild animals in the Orders Carnivora and Artiodactyla held by members of
the National Federation of Zoologcal Gardens of Great Britain and Ireland on December 31, 1989 (Bennett 1990) and the
population at that time of the species in which spongifonn encephalopathies have been diagnosed.
Tribe/ Number of
Order subfamily Species species Individuals
Artiodactyla 62 2675
Strepciserotini 14 304
Taurotragus oryx 35
Tragelaphus angasi 26
Tragelaphus strepsiceros 17
Hippotragini 10 270
Oryx dammah 65
Oryx gazella 29
Oryx leucoryx 5
Carnivora 69 1227
Felidae 23 543
Felis concolor 16
Acinonyx jubatus 42

NEWS AND REPORTS

THE VETERINARY RECORD SEPTEMBER 24,1994 VOL 135 PAGES 294-295

ANIMAL HEALTH
INTERNATIONAL DISEASE SURVEILLANCE: APRIL TO JUNE 1994

THE following report is compiled from information received by the Ministry of Agriculture, Fisheries and Food, Tolwolth,
Surrey, from international disease reporting agencies and government veterinary sources. It should be noted that some of
the information is not received until a considerable time after the occurrence of disease and that the absence of a report
does not necessarily mean the absence of disease.

AFRICAN SWINE FEVER

ITALY The disease remains confined to the eastern region of Nuoro in Sardinia totalling 80 outbreaks so far in 1994. This
is a significant increase on the 54 outbreaks reported during the first six months of 1993. A total of 96 outbreaks were
reported in 1993.
SPAIN No further outbreaks were reported during the second quarter of 1994 so the total remains at 11. This is a
substantial reduction on the 29 outbreaks reported during the same period in 1993.

GERMANY A further 48 outbreaks were reported making a total of 78 in 1994. A total of 99 outbreaks were reported in
1993.

ITALY Ten further outbreaks were reported to bring the total to 12 for 1994, all in the previously affected regions of
Nuoro (Sardinia) except one, on May 24, at Sassari which is north of Nuoro.

BELGIUM Outbreaks continued to occur in Belgium, with a further 35 being reported, bringing the total to 44 in 1994.
All were in Oost Vlaanderen except one in West Vlaanderen on June 8. A total of seven outbreaks occurred in 1993.

BULGARIA No further outbreaks have occurred since five were reported between December 16, 1993, and March 28,
1994.

ESTONIA No further outbreaks have occurred since one on January 15.

SLOVAK REPUBLIC. No further outbreaks have occurred since 10 were reported between December 7 and January 26.

POLAND The first outbreaks in 1994 were reported on four small mixed units on April 8 in the Zamosc province, in the
eastern part of the country. A fifth outbreak was reported involving one sow and six weaners on July 7 in the Gorzow
Wiekopolski province in the western part of the country. The origin of all five outbreaks was suspected to be the illegal
importation of fresh meat.

YUGOSLAVIA Two further outbreaks were reported in adjoining small private farms on April 4, both of which occurred
in the central Banat district of the Serbian Republic. A total of six have now been reported in 1994. The origin of infection
is still unknown.

MACEDONIA (formerly part of the republic of Yugoslavia) The first outbreak in 1994 was reported on a collective farm
involving 19, in the Stip municipality, in the eastern part of the country.

CZECH REPUBLIC The first outbreak in 1994 was reported on a unit of 2523 fattening pigs on May 4 in the Tabor
district (south Bohemia region).

Swine vesicular disease

ITALY In the second quarter of 1994, Italy reported a further 11 outbreaks, which were scattered throughout the regions of
Bergamo (one), Cosenza (one), Cremona (one), Mantova (one), Matera (one), Modena (two), Nuoro (one) and Palma
(three). A total of 24 have occurred so far in 1994.

NETHERLANDS No further outbreaks have occurred in the Netherlands since February 28 in Drenthe.

Contagious bovine pleuropneumonia

ITALY No outbreaks have been reported in Italy during 1994. A total of 10 outbreaks were reported in 1993, which
represented a significant decrease from the 34 in 1992.

SPAIN One further outbreak in which nine of 30 cattle were affected occurred in April in Gulpazcoa (Basque country).
The outbreak is thought to be linked to a purchased animal from the second of the two previous outbreaks which occurred
in Cantabria in February 1994.

PORTUGAL The disease remains established in the previously affected north west area of the country, namely Aveiro,
Bragu and Porto, and there were also outbreaks in Guarda in the north east, Beja in the south and Itha da Terceira in the
Azores. The total of 31 outbreaks so far in 1994 is a substantial reduction on the 332 outbreaks reported during the same
period in 1993.

Bovine spongiform encephalopathy

FRANCE In France three cases of bovine spongiform encephalopathy were reported in the Brittany region. The first was
in March and the animal concerned had been born in January 1989 and had remained in the herd of origin. The two cases
were reported in May 1994. They had both been born in December 1988 and had remained in the same herd of origin.
GERMANY A further case of BSE was confirmed on May 26. The eight-year-old Welsh black cow had been imported
from Great Britain into Germany in 1988.

Foot-and-mouth disease

TURKEY During January to March 1994 inclusive there were 101 outbreaks in Turkey caused by type O virus.

ALGERIA AND MOROCCO There were no outbreaks reported during the period January to March.

Newcastle disease

BELGIUM In Belgium, one outbreak was reported in Antwerpen on June 3, in a consignment of psittacine birds in
quarantine reported in Antwerpen on June 3, in a consignment of psittacine birds in quarantine after importation from
Asia. No other outbreaks have been reported in 1994.

GERMANY In Germany, a further 72 outbreaks were reported during the second quarter of 1994 to bring the total to 76
for 1994. The lander affected included Brandenburg, Mecklenburg Vorpommern, Nordrhein, Rhineland-Palatinate,
Saxony, Schleswig-Holstein, Thuringen and Westphalerland. This is a significant increase over the total of 44 outbreaks
reported during 1993 and four for the first quarter of 1994.

NETHERLANDS A further four outbreaks were reported, two in Utrecht and two in Ijsselmeerpolders. A total of six
outbreaks have been reported so far in 1994. A total of 21 outbreaks were reported in 1993. A variety of types of birds have
been atfected in the recent outbreaks including a large broiler unit hobby fowl and pigeons imported from Asia.

PORTUGAL A further two outbreaks were reported in the second quarter of 1994, all again in Viseu, in the north west of
the country, to bring the total to six for 1994.

ITALY Four outbreaks were reported, in Pavia, Chieti, Roma and Savona.

SWITZERLAND No further outbreaks have been reported since the one in the Bern canton on February 15 involving six
birds.

SOUTH AFRICA Two further outbreaks were reported, again affecting ostriches, on May 16 in the Transvaal province,
making a total of three in 1994.

ZIMBABWE The first outbreak was reported in June 6 in backyard rural chickens in the Ndanga communal area. A
further outbreak was reported in the Masvingo province on June 23. The two outbreaks were associated with the
movement of poultry.

Fowl plague

NETHERLANDS An outbreak was reported on April 7 in the province of North Brabant affecting 193 birds (138 emus,
41 nandus, 11 cassowaries and three crowned cranes). This was later found to be an incorrect laboratory diagnosis as the
strain of virus isolated was found to be non-pathogenic. The birds had already been slaughtered. The Netherlands
therefore remains free from fowl plague.

Fowl influenza

SOUTH AFRICA A single outbreak was reported on May 16 in ostriches in the Western Cape province. The origin is
unknown.

Products and Services

Two new avermectins for use in cattle

TWO companies have introduced broad-spectrum parasiticides for use in cattle recently. In August, Janssen Animal
Health introduced Enzec Injection for Cattle, containing abamectin; earlier this month, Pfizer Animal Health introduced
Dectomax injectable solution, containing doramectin. Both compounds belong to the avermectin class of parasiticides, of
which ivermectin is another example.

Enzec
Enzec injection, from Janssen, contains 1 per cent w/v abamectin and is indicated for the treatment and control of
gastrointestinal nematodes, lungworms, warbles, mange mites and sucking lice of cattle.

Dosage and administration.- 1 ml (10 mg abamectin) per 50 kg bodyweight, based on a recommended dose of 200 ug
abamectin per kg bodyweight, injected subcutaneously.

Contraindications, warnings, etc.- Do not treat calves under 16 weeks of age, or use in cattle producing milk for human
consumption. Animals must not be slaughtered for human consumption during treatment, or within 42 days of last
treatment.

Legal category.- PML.

Packaging.- 50 ml, 200 ml and 500 ml containers.

Product licence holder.- Merck SharP & Dohme Ltd, Hertford Road, Hoddesdon, Hertfordshire EN11 9BU.

Distributor.- Janssen Animal Health, Grove, Wantage, Oxfordshire OX12 ODQ.

Dectomax
Dectomax injectable solution, from Pfizer, contains 1 per cent w/v doramectin and is indicated for the treatment and
control of gastrointestinal roundworms, lungworms, eyeworms, warbles, lice and mange mites. It may also be used as an
aid in the control of Nematodirus helvetianus and of biting lice (Damalinia bovis).

Dosage and administration.- The recommended dosage of 200 ug doramectin per kg bodyweight should be given by
subcutaneous injection at the rate of I ml/50 kg. It is recommended that cattle which are set-stocked are treated at turn out
and eight weeks later. This injection programme is claimed to allow the development of natural immunity to lung worm.

Contraindications, warnings, etc.- Animal must not be slaughtered for human consumption during treatment or within 42
days of the last treatment. The product should not be used in cows producing milk for human consumption, or in non-
lactating dairy cow including pregnant dairy heifers.

Legal category.- POM.

Packaging.- Multi-dose vials of 50 ml, 200ml and 500 ml.

Product licence holder.- Pfizer Animal Health, Sandwich. Kent CT13 9NJ.

ABSTRACTS
THE VETERINARY RECORD SEPTEMBER 24,1994 VOL 135 PAGE 303

Isolation of Listeria species from broilers

SAMPLES of skin from the necks of broilers slaughtered at two processing plants (A and B) were examined for the
presence of Listeria species, first after they had left the final rinser but before entering the chiller, and again immediately
after they had left the chiller, at plant A the chiller contained water with from 2 to 15 ppm free chlorine and at plant B it
contained water with approximately 10 ppm chlorine.

Listeria monocytogenes was isolated only from the broilers sampled at plant A after chilling (58 per cent of 62 samples);
at plant A L innocua was isolated from 19 per cent of the samples taken before chilling and from 3 per cent of those taken
after chilling, and at plant B this organism was isolated from 39 per cent of the samples taken before chilling and from 6
per cent of those taken after chilling. The frequency of isolation of L monocytogenes at plant A may have been due to the
occasionally inadequate concentration of available chlorine present in the chilling water.

LONCAREVIC, S., THAM, W. & DANlELSSON-THAM, M.-L.(1994) Acta Veterinaria Scandinavica 35, 149 Acta
Veterinaria Scandinavica 35, 149

Cryptosporidiosis in dairy calves

CRYPTOSPORIDIOSIS is a parasitic disease of calves, transmitted by the faecal-oral route, which usually causes a
transient mild to severe diarrhoea. However, in immunosuppressed people cryptosporiosis is a serious disease, and it can
be contracted from infected cattle. A monoclonal antibody technique was used in a nationwide survey of 7369 faecal
samples taken from calves on 1103 farms.

Oocysts of cryptosporidia were found in samples from the calves on 652 (59-1 per cent) of the farms, and in the samples
from 1648 of the calves (22-4 per cent). Almost half the calves aged seven to 21 days had cryptosporidia in their faecal
samples. The prevalence of cryptosporidia was highest in the summer, and farms with more than 100 milking cows and
farms where the cows did not calve in individual boxes were significantly more likely to have calves with cryptosporidia.

GARBER L.P.,SALMAN, M.D.,HURD,H.S.,KEEFE,T. & SCHLATER J.L.(1994) Journal of the American Veterinary
Medical Association 205,86

Danger of misassembly of anaesthetic apparatus

TWO horses which were being anaesthetised for orthopaedic surgery suddenly deteriorated in cardiovascular and
respiratory status and lost their ocular reflexes after being moved to the operating table. An investigation revealed that the
problem was due to a mistake in the connection of the Matrix Large Animal Circle anaesthetic machine to the Fluotec
Mark III vaporiser, as a result of which the flow of fresh gas through the vaporiser was reversed. Experimental tests
showed that when the flow of gas was reversed, the concentration of halothane delivered was increased by approximately
30 per cent at a flow rate of 1 litre/minute, by 60 per cent at 4 litres/minute and that it was more than doubled at a flow
rate of 8 litres/minute. The authors suggest that the design of the equipment should be modified to make it impossible for
it to be assembled incorrectly.

GREGG,A.S.,JONES,RS.&SNOWDON,S.L.(1993) British Journal of Anaesthesia 71, 303

PAPERS AND ARTICLES

THE VETERINARY RECORD SEPTEMBER 24,1994 VOL 135 PAGES 294-295

Development of early bovine embryos in different culture systems

H.L.Feng,Q.Z.Yang,Q.Y.Sun,P.C.Qin,J.M.Liu

The objective of this study was to examine the effects of different culture systems on the development of early bovine
embryos in vitro. A total of 1089 oocytes were aspirated from 2 to 5 mm follicles of ovaries collected at a local abattoir; a
high proportion of the oocytes matured in vitro were fertilised by spermatozoa capacitated with caffeine and heparin.
Seven to eight hours after insemination, the oocytes were transferred into three in vitro systems: A, TCM 199 + 10 per
cent fetal calf serum culture medium, B, coculture with a monolayer of granulosa cells and C, coculture with bovine
oviductal epithelial cells. The results showed that the proportion of the early bovine embryos which overcame the block at
eight to 16 cells and developed to the morula and blastocyst stages in system C was significantly higher than in systems A
or B.

SINCE the first in vitro fertilised (IVF) calf was born in the early 1980s (Brackett and others 1982), the methods for in
vitro maturation and fertilisation have improved markedly. Bovine morulae and blastocysts can now be obtained routinely
from oocytes collected from slaughterhouse ovaries and matured and fertilised in vitro. Although pregnancies and live
calves have been obtained from the transfer of embryos developed in vivo in many countries (Goto and others 1988, Lu
and others 1988, Jiang and others 1991, Monson and others 1992, Yang and others 1992, Zhang and others 1993), the
rate of success has been lower when the embryos have been cultured in vitro, because of a block in their development at
the eight to 16 cell stage. The rate of development of the resulting embryos was usually slower than that of normal
embryos in vivo, the proportion of the embryos which reached the blastocyst stage was low and few survived to term (Xu
and others 1992). Further improvements are therefore needed if the in vitro ma turation and fertilisation of oocytes and
the in vitro culture of the resulting embryos are to be used reliably for research and commercial purposes.

The present study was carried out to evaluate the effects of three in vitro systems on the development of early bovine
embryos and to try to establish the optimum in vitro culture system.

MATERIALS AND METHODS

Oocyte collection and maturation in vitro

Ovaries obtained at an abattoir within 15 minutes of slaughter were placed in a thermos flask at 32 to 36°C, containing
normal saline with penicillin G (400 iulml) and streptomycin (500 llg/ml). The ovaries were transferred to the laboratory
within one to three hours where they were immediately washed at 32 to 36°C in saline containing antibiotics. The oocytes
were aspirated with an 18 gauge needle from follicles 2 to 5 mm in diameter and placed into 60 x 15 mm petri dishes
(Falco Plastics 1007). The oocytes were recovered from the settled follicular fluid in the dishes by using a
stereomicroscope. Only oocytes with an intact, compact cumulus oophorus and evenly granulated cytoplasm were
cultured. Selected oocytes were washed twice in carbon dioxide-equilibrated maturation medium before the final in vitro
maturation. The oocytes were randomly assigned to 100 ul droplets of TCM 199 (Gibco) + 10 per cent fetal calf serum (10
to 20 cells/drop) for maturation under sterile paraffin oil in a humidified atmosphere of 5 per cent carbon dioxide in air at
38 5°C for 20 to 24 hours.

Establishment of coculture systems

Bovine oviducts that were ipsilateral to ovaries containing a corpus haemorrhagicum were collected at a local abattoir and
placed in 50 ml conical tubes containing phosphate buffered saline (PBS) supplemented with 100 iu penicillin and 100
@lg streptomycin/ml. The tubes containing the oviducts were placed in a thermos flask containing ice until they were
processed at the laboratory. The oviductal cells were collected by agitating the oviducts in 0 76 per cent EDTA/PBS; the
oviducts were trimmed free of connective tissue and rinsed in PBS; after cutting off the fimbrial and thermos fiask
containing ice until they were processed at the laboratory. The oviductal cells were collected by agitating the oviducts in
0.76 per cent EDTA/PBS; the oviducts were trimmed free of connective tissue and rinsed in PBS; after cutting off the
fimbrial and utero-tubal junctions, the oviduct was cut open longitudinally with sterile scissors and cut into sections
approximately 10 mm long, which were rinsed three times with pre-incubated EDTA/PBS and then incubated in 35 x 10
mm petri dishes containing 0.76 per cent EDTA/PBS at 38.5°C for two hours. After incubation, the epithelial sheets or
oviductal cell pellets were extracted from the lumen into a 35 mm petri dish by means of small sterile forceps. The clumps
of cells were further broken down by repeated aspiration through an 18 gauge needle attached to a 1-0 ml syringe
containing 1 ml of EDTA/PBS. The resulting suspension of clumps of epithelial cells was transferred to a 15 ml conical
tube containing 10 ml of TCM 199 and washed by centrifugation. The cells were resuspended in fresh TCM 199 and
washed two or three more times. Afer washing, the cell pellet was resuspended in TCM 199 + 10 per cent FCS (v/v)
suplemented with 100 iu of penicillin and 100 ug of streptomycin/ml. This cell isolation technique provided sufficient
cells from a single oviduct to seed four-well tissue culture plates (Swedesboro) which were cultured at 38 5°C in 5 per cent
carbon dioxide in humidified air. Thirty-six to 48 hours after the start of the culture, the medium and unattached cells
were removed, and 0.4 to 0.5 ml of fresh TCM 199 + 10 per cent FCS was added. Confluent monolayers with some ciliary
activity formed within three to five days and were used for coculture.

Granulosa cells for coculture were collected from the follicular aspirates. These cells were pooled and washed three times
in PBS. The cells were then dispersed mechanically by pipetting them repeatedly with a 1 ml pipette until all the cell
clumps had been reduced to a suspension of single cells. This cell suspension was washed once more with PBS, and the
granulosa cells were resuspended in the oviductual cell culture medium and dispensed directly into four-well culture
plates two to three days before they were used.

In another method for culturing the granulosa cells, the proliferating monolayer of cells from the original oocytes duling
culture were left as they were. New medium was added to the droplet at intervals of 36 to 48 hours.

IN VITRO FERTILISATION

The oocytes cultured for 20 to 24 hours were washed three times in BO liquid with bovine serum albumin BO (20 mg/ml)
and heparin (20 ug/ml) and then placed in droplets (final volume 100 ul) of BO liquid at 10 oocytes per droplet.

TABLE 1: The fertilisation and development of in vitro fertilised bovine ova in different culture systems

Number of Number (%) of embryos

Culture Number oocytes cultured (%) developed invitro
system of trials in vitro fertilised 24 cell 8-16 cell M and sLA

466 (86) 295 (63 3) 158 (53 6) 73 (24 7)b

300 (95) 209 (69-7) 116 (55 5) 60 (28 7)b
224 (97) 149 (66 5) 87 (58-4) 78 (52 3)C

M Morula, BL Blastocyst
* Percentages with different superscripts differ, P<0.01
Frozen bovine semen was used to fertilise the oocytes. The spermatozoa were washed twice in sperm BO liquid,
supplemented with 10mM caffeine, by centrifugation at 833 g for five minutes. The dense sperm suspension (100 ul) was
added to each droplet of BO liquid containing oocytes; the final concentration of spermatozoa was 2 to 4 x 10 6 cells/ml.
The conditions for the coculture of sperm and oocytes were the same as those used for the maturation of the oocytes (Yang
and others 1991).

IN VITRO CULTURE

Seven to eight hours after insemination, most of the cumulus cells were removed mechanically from the oocytes by using
a finely drawn fire-polished pipette in a washing process. The fertilised oocytes were either transferred into droplets (10 to
15/drop) of TCM 199 + 10 cent per FCS (system A) or cocultured with a monolayer of granulosa cells (system B) or
cocultured with bovine oviductal epithelial cells (system C) at 38 5°C in 5 per cent carbon dioxide in air and 100 per cent
humidity. Each medium was replaced at 36 to 48 hour intervals. Between days 7 and 8, the embryos were scored for their
development to morulae and blastocysts. The embryos were examined by phase-contrast microscopy and those showing
compaction were classified as morulae and those with a blastocoele cavity were classified as blastocysts.

STATISTICAL ANALYSIS

The significance of differences between the treatment groups was established by chi-square analysis. Significance was
accepted if P<0.05.

RESULTS

Characteristics of granulosa and oviductal cell cultures

The oviductal epithelial and granulosa cells were observed to be quite different when placed in culture. The oviductal,
cells were composed of small clumps of spherical, ciliated cells which, over the initial 24 hours in culture, either
dispersed into single cells or formed ciliated vesicles that never became attached. Attachment occurred to only a small
extent during this initial period of 24 hours. During the second period of 24 hours, larger groups of epithelial cells were
observed to attach and spread over the plastic surface of the wells, resulting in the formation of a 50 to 60 per ccnt
monolayer. After 72 hours in culture, the oviductal monolayers could be distinguished into two morphologically distinct
cell populations, consisting of islands of small, oblong, ciliated cells separated by tracts of more slender secretory type
cells. By contrast, the granulosa cells consisted of morphologically homogeneous squamous-type cells which progressed to
form a 40 to 50 per cent monolayer after 48 hours in culture.

Development offertilised eggs in vitro

The results are summarised in Table 1. The proportions of the fertilised eggs which developed from the two-cell to the 16-
cell stage did not differ significantly between the three cultivation systems. However, in system C a higher proportion of
the fertilised eggs developed to the morula and blastocyst stages than in either system A or system B.

DISCUSSION

The high fertilisation rate achieved confirmed that sperm treated with heparin and caffeine can fertilise oocytes matured
in vitro effectively (Yang and others 1991, 1992).

The results also clearly indicated that oocytes retrieved from a slaughterhouse and matured, fertilised and cultured in vitro
can develop to the morula and blastocyst stages. Although the proportions of embryos developing to the eight to 16-cell
stage were not significantly different between the three culture systems, more of the embryos cultured in system C
developed beyond the 16-cell stage to become morulae and blastocysts. Kajihara and others (1987) reported that in the
culture of bovine eggs matured and fertilised in vitro, the cumulus cell monolayer was essential for their development to
the blastocyst stage.

Fukuda and others (1990) suggested that the cumulus cell monolayer was unable to promote the development of embryos
for more than 96 hours after insemination, but that the gulosa cell monolayer produced factors with beneficial effects on
in vitro development (Goto and others 1988). The present study indicated that coculture with bovine oviductal epithelial
cells (system C) was more efficient than coculture with bovine granulosa cells (system B) in enhancing the development
of embryos to the morula and blastocyst stages. It is possible that system C was more effective because of cell to cell
interactions within the clusters of oviduct cells. Recently, it has been demonstrated that monolayers of oviductal cells
enhanced the in vitro development of early stage embryos in sheep (Rexroad and Owell 1991), cattle (Eyestone and First
1989, Ellington and others 1990), pigs (White and others 1989), goat (Sakkas and others 1989), horses (Ball and
Alfschall 1990) and humans (Sathananthan and others 1990). These reports further suggested that the oviductal cells can
secrete embryotrophic factors, and that the use of serum-free medium does not alter the secretory capacity of the oviductal
cells during coculture (Rexroad and Owell 1991). It has been proposed that the embryotrophic effects of coculture with
oviductal cells may be due to the ability of the oviductal cells to maintain a required minimal rate of protein secretion
(Gandolfi and others 1989). Although monolayers of both oviductal epithelial cells and granulosa cells can secrete
embryonic factor(s), it is possible that the different cell types could produce different beneficial factors, or different
amounts of the same factor. In addition, bovine oviductal epithelial cells may promote the metabolism and development of
the embryo by means of their ciliary action and this may be the reason why system C was more efficient than the other
two systems of embryo culture.

An efficient and reliable in vitro coculture system for bovine embryos has been established, using bovine oviductal
epithelial cells. The system could be useful for basic and applied studies, because most aspects of embryo development
examined were comparable to those in vivo. A limitation of the system at present is that it requires the collection of fresh
oviducts each time to set up the primary culture of the oviductal cells; this is inconvenient and a source of uncontrollable
variation. The development of methods for maintaining the normal function of the cells, or for cryopreserving the cells,
may overcome these problems.

SHORT COMMUNICATIONS
THE VETERINARY RECORD SEPTEMBER 24, 1994 VOL 135 PAGES 306-307

Use of intraocular adrenaline during cataract extractions in dogs

S. M. Petersen-Jones, R. E. Clutton

INTRAOCULAR adrenaline may be used during cataract surgery in dogs to achieve and maintain mydriasis. However,
the arrhythmogenicity of adrenaline in people and dogs is potentiated by volatile anaesthetics with halothane being more
arrhythmogenic than enflurane or isoflurane (Johnston and others 1976). For this reason the intraocular application of
adrenaline in halothane-anaesthetised animals is often considered imprudent. Rubin (1968), for example, reported that
intraocular lavage with 100 ug/ml of adrenaline was associated with ventricular fibrillation in two of his canine patients
and was fatal in one of these cases and Slatter (1990) states that there is a significant risk of ventricular fibrillation when
intraocular adrenaline (100 ug/ml solution) is used in halothane-anaesthetised dogs.

Studies in man, however, have shown that intraocular adrenaline can be used safely. Smith and others (1972) monitored
the electrocardiogram (ECG) of human patients undergoing cataract extraction under halothane anaesthesia, following
pre-medication with pentobarbitone and atropine in children and hydroxyzine, pentobarbitone and atropine in adults, and
found no alterations with intraocular injections of adrenaline ranging from 40 to 700 ug (0.4 to 68.0 ug/kg). Fell and
others (1989) showed that the use of an intraocular irrigation solution containing 2 ug/ml adrenaline did not alter plasma
levels of adrenaline or noradrenaline. Similar investigations have not been reported in dogs. To investigate whether
intraocular administration of a mydriatic dose of adrenaline is associated with significant cardiac side effects in
halothane-anaesthetised dogs, the ECGS of 40 dogs undergoing routine extracapsular cataract extraction were monitored.

Forty dogs of a variety of breeds ranging in age from two to 15 years and weighing from 4 kg to 36.5 kg were scheduled
for routine extracapsular cataract extraction. Three dogs were suffering from diabetes mellitus which was adequately
controlled by insulin therapy while the rest were clinically normal, apart from having cataracts. Pre-anaesthetic
medication in all cases was acepromazine maleate (ACP; C-Vet) 0.05 mg/kg and buprenorphine (Temgesic; Reckitt &
Colman) 10 ug/kg, given by slow intravenous injection. Twenty minutes later the cephalic vein was catheterised and
anaesthesia induced in 27 of the dogs with 2.5 per cent thiopentone (Intraval sodium; Rhane Merieux) at 6 to 8 mg/kg
intravenously. The remaining 13 received propofol (Rapinovet; Pitman-Moore) 4 mg/kg intravenously.

The trachea was intubated with a cuffed endotracheal tube which was connected to an appropriate anaesthetic breathing
system (Mapleson A, D, F or circle). Anaesthesia was maintained with halothane delivered from a vaporiser (Fluotec Mk
III) in an oxygen/nitrous oxide (1:2) mixture. The initial delivered concentration was set at 15 per cent; 1 per cent was
delivered during surgery. An intravenous infusion of Hartmann's solution (10 ml/kg/hour) was given during anaesthesia.
Just before surgery neuromuscular block was achieved with vecuronium bromide at 50 ug/kg intravenously (Norcuron;
Organon Laboratories) and the dogs were ventilated mechanically (Manley Pulmovent Model MPP) with 1 per cent
halothane carried in a mixture of oxygen/nitrous oxide (total fresh gas flow rate: 300 ml/kg/min) and carbon dioxide (12
ml/kg/min). Throughout anaesthesia vital signs were monitored, heart and lung sounds were auscultated with an
oesophageal stethoscope and the ECG was recorded (lead II). Body temperature was supported using heating blankets and
insulation.

Additional pre-operative medication consisted of dexamethasone sodium phosphate (Dexadreson; Intervet UK) at 2 mg/kg
and flunixin meglumine (Finadyne; Schering-Plough) at 0 5 mg/kg by slow intravenous injection and cimetidine
(Tagamet, SmithKline Beecham) at 5 mg/kg intramuscularly. Diabetic dogs were given methylprednisolone sodium
succinate (Solu-Medrone V; Upjohn) at 10 mg/kg instead of dexamethasone.

Following preparation and positioning the anterior chamber was penetrated via a clear corneal incision adjacent to the
limbus and 100 ug of adrenaline (1 mg/ml adrenaline; Martindale), the equivalent of between 2.7 and 25 ug/kg, was
injected into the anterior chamber via a 26 gauge anterior chamber cannula. In all eyes this rapidly resulted in acceptable
mydriasis. Following delivery of the cataractous lens nucleus, residual cortical material was aspirated using an
irrigating/aspirating cannula with balanced salt solution (Alcon Laboratories) containing 2 llg@ml adrenaline and 10
iu/ml heparin sodium (Leo Laboratories) as the irrigating solution. This solution was also used to reform the anterior
chamber. The volume of irrigating solution used was variable and was not recorded. In 22 of the patients a unilateral
cataract extraction was performed and in 18 a bilateral procedure was carried out under the same anaesthetic.

The heart rate did not change, nor were arrhythmias seen within 10 minutes of the initial injection of adrenaline into the
anterior chamber. Indeed, arrhythmias, including sinus tachycardia, were not observed during surgery in any animal in
the study. Monitoring of the dogs for two months postoperatively did not reveal any ocular changes attributable to the use
of adrenaline.

The dose of adrenaline which causes arrhythmias is lowered by halothane in people (Johnston and others 1976) and dogs
(Muir and others 1975), an effect which is independent of halothane concentration (Metz and Maze 1985). The resulting
arrhythmias are of particular concern because they tend to be malignant. Various mechanisms for this effect have been
postulated with recent attention directed towards cardiac alpha-2 receptors (Spiss and others 1984, Dresel 1985).
Conversely, halothane can also have anti-arrhythmic effects under specific circumstances (Muir and others 1988). The
duality of halothane arrhythmogenicity is probably influenced by prevailing physiological conditions and other drugs
present; thiobarbiturates and alpha-2 agonists predispose to arrhythmias in dogs (Bednarski and others 1985).

Currently it is felt that halothane per se is unlikely to cause ventricular or supraventricular tachydysrhythmias in the
absence of pathophysiological factors such as disease states, catecholamine excess, digitalis excess, hypoxia or myocardial
infarction (Atlee 1985). In the present study, with the exception of adrenaline administration, arrhythmogenic risk factors
were minimised. In particular, all dogs received acepromazine maleate, which has a demonstrable anti-arryhthmic effect
with regard to ventricular arrhythmias and fibrillation in dogs anaesthetised with halothane (Muir and others 1975). No
cases involved were receiving digitalis glycosides or other cardioactive drugs.

Because neuromuscular blockers were used, ventilation was mechanically controlled, reducing the risk of hypercapnia
(Paco2> 5 85 kPa[44 mmHg]) and, or hypoxia (Pao2<7 97 kPa[60mmHg]). Neither end-tidal carbon dioxide nor oxygen
were measured in this series although the ventilation regime imposed normally produces Paco2 values from 425 to 651
kPa (32 to 49 mmHg). Particular attention was paid to the adequacy of anaesthesia. Circulating blood volume was
supported by intravenous fluid infusion and body temperature maintained using heating blankets and insulation. In
diabetic animals, steps were taken to ensure normoglycaemia throughout surgery.

Intraocularly administered adrenaline could be systemically absorbed via the aqueous drainage pathways, through the
anterior uveal vasculature or across the conjunctiva after leaking out of the eye. The lack of any detectable ECG changes
associatcd with the use of adrenaline at these dose rates suggest that in the dog, as has already been shown for humans,
there is a low level of systemic absorption from the anterior chamber. Adrenaline has a vasoconstrictive action which
would be expected to reduce its systemic absorption via conjunctival and anterior uveal vessels, but it also acts to increage
aqueous outflow which would tend to have the opposite effect.

This study showed that the intraocular administration of adrenaline at sufficient levels to achieve and maintain mydriasis
in dogs anaesthetised with halothane following acepromazine and buprenorphine premedication was not associated with
an increase in heart rate, or any arrhythmias. Furthermore, ocular pathology associated with its use could not be clinically
detected. The authors therefore conclude that the use of intraocular adrenaline following the protocol outlined above is
safe