0 The second lesser pathway was from the aortic bulb to the brachiocephalic artery, to the right costocervical artery, to the costal branch of the right costocervical a., to the right seventh dorsal intercostal a. and then to the thoracic aorta.

The neurological findings may be explained as the result of pressure and displacement effects of the pulsating anastomosis of the enlarged left and right vertebral arteries on the cervical spinal cord. The cavitation of the second and third cervical vertebrae may also be explained in terms of a similar pressure effect on the vertebrae during foetal growth and development.

The abnormal arterial vascular pattern found in this dog was consistent with what may be anticipated if the left fourth aortic arch failed to develop (Figure 4) and the right fourth aortic arch failed to develop or persist. The remarkable aspect of this case was that the dog was able to survive to 5 months of age in the absence of any direct connection between the heart and thoracic aorta.

i - cc

.b

I X

Figure 4a Figure 4b

Figure 4a. Early embryonic aortic arch pattern.

Figure 4b. Postnatal pattern of the great arteries. Shaded area represents the left fourth aortic arch artery (4A) and its con- tribution to the postnatal great artery pattern (46). Modified from Moore (1982).

The authors wish to thank Dr H Bruhl who referred the case to the University of Sydney Veterinary Clinic for further evaluation and Ms B Jantulik who prepared the artwork for the figures.

Legend to Figures a Aortic bulb b Thoracic aorta c Ligamentum arteriosum cc Ductus arteriosus cl . c2. c3 and c4 Cervical vertebrae one to four d e f 9 h i j k I m n

P 9 r

t

0

S

U V W X

Y

Left vertebral artery Right vertebral artery Left seventh dorsal intercostal artery Right seventh dorsal intercostal artery Left subclavian artery Right subclavian artery Left internal thoracic artery Right internal thoracic artery Pulmonary trunk Oesophagus Trachea Pulmonary vein Heart Rib (2,4,6 and 8 ) Azygos vein Cranial vena cava Right costocervical artery, costal branch Right costocervical artery, cervical branch Brachiocephalic artery Left common cartoid artery Riaht common carotid artery Transverse foramen

1, 2, 3, 4, 5 and 6 Aortic arch arteries one to six.

220

References Moore K L (1982) - The Developing Human: Clinically Oriented

Embryology, 3rd edn Saunders, Philadelphia.

(Accepted for publication 5 March 1987)

Detection of antibody to canine parvovirus in dog sera by enzyme immunoassay

Graduate School of Tropical

James Cook University of

Townsville, Queensland 4810

A E TABOR Veterinary Science, R M PATTERSON*

J R SMITH North Queensland, K G SUARYANA

G W BURGESS

Canine parvovirus (CPV) is a highly contagious infectious agent of acute gastroenteritis, which in non-immune dogs often causes death (Johnson and Spadbrow 1979). Antibody to CPV in dog serum is commonly quantitated by the haemagglutin- ation inhibition assay (HAI). However, the HA1 assay requires initial absorption of serum to remove non-specific agglutinins and inhibitors, which can reduce specific HA1 titres by up to 4 foid (Carmichael et a1 1983). It is also time consuming and requires laboratory facilities.

The response to vaccination of pups against parvovirus infection can be interfered with by existing maternal antibody if titres exceed 8 HA1 units (Smith 1986). Thus timing of vaccination is critical for maximum efficacy to be achieved, hence there is a need for a rapid accurate means of determining protective antibody titres to CPV.

This report describes a sensitive and rapid enzyme-linked immunosorbent assay @LISA) for the detection of antibody to CPV in dogs.

ELISA antigen was produced from CPV (K31, R H John- son) infected feline kidney (FK) cells (R H Johnson unpub- lished) maintained in Eagle’s basal medium with Hank’s so- lution, 10% foetal calf serum and antibiotics. Four-to-five- day infected cell monolayers were frozen and thawed, followed by inactivation of the virus with 0.2% &propiolactone at 4OC overnight. Viral preparation was then clarified of cell debris by centrifugation at lOOOg for 10 min and the viral antigen preparation in the supernate concentrated by stirred cell ul- trafdtrationt. The viral suspension was precipitated with an equal volume of saturated ammonium sulphate and then sub- jected to successive treatments with 0.1070 sarkosyl with gentle sonication. Virus was then pelleted at 100,OOO g for 2 h in a Beckman L5-75B ultra centrifuge. The viral pellet was then re-suspended in phosphate buffered saline (PBS) and further purified through a 40% sucrose cushion. The viral pellet was finally resuspended in .PBS, aliquoted and stored at - 2OoC. A control cell antigen was prepared from uninfected FK cell monolayers similarly to the CPV antigen with the sarkosyl and sucrose cushion purification steps omitted.

Viral or cell culture antigen was absorbed to wells of ac- tivated polyvinyl chloride immuno assay plates by incubation of 2 pg/ml of antigen in a bicarbonate buffer, pH 9.6, at room temperature for 16 h. Plates were then washed in PBS containing 0.05% tween-20 in a microplate washer$ or by hand with a wash bottle. Plates were stored at 4OC until use. Serums from 70 dogs of known vaccination history were

used to evaluate the ELISA. They were divided into 3 cate- gories: those from unvaccinated dogs, those from dogs vac- cinated with CPV/distemper/hepatitis (CPV/D/H) and those from dogs which had received distemper/hepatitis (D/H) vac-

Address for correspondence t Amicon 8400, with Diaflow Ultrafilter XM100 3 Titertek Multiscan MCC, Flow Laboratories $ Multiscan MCC, Titertek, Flow Laboratories

Australian Veterinary Journal, Vol. 64. N O . 7, July, 1987

150 17ot

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HA1 TlTRE Figure 1 . Distribution of ELISA values (O.D.) and HA1 titres for the 3 groups of dog sera. 0 Unvaccinated, n = l 4 ; 0 CPVl distemper/hepatitis vaccinated, n=32 and A Distemper/hep- atitis vaccinated only, n=24.

cine but not CPV vaccine. These serums varied over a wide range of HA1 activity. Each serum was diluted 1: 100 in TEN- {ween buffer (150 mM tris, 0.1 mM EDTA, 150 mM NaCl + .05% tween-20) and incubated 50 pl/well for 1 h at room

I emperature in antigen coated plates, after which time plates were washed.

A rabbit anti-dog IgG-horse-radish peroxidase conjugate (Miles-Yeda) was used at 1:lOOO dilution in TEN-tween to detect dog antibodies. Diluted conjugate was added 50 pl/ well and incubated for a further 1 h, after which plates were again washed and reacted with 100 pl/well 52mM 2.2' azino- di-(3-ethyl benthiazoline sulfonic acid) with 130 mM hydrogen peroxide in a citrate/phosphate buffer, pH 4.2. After 1 h plates were read spectrophotometrically at 414 and 492 nm using a micro plate reader$ interfaced to an Apple IIE mi- crocomputer.

HA1 assays were performed according to previously de- scribed techniques (Joo et al 1976) and a parametric correlation co-efficient was used to determine significant correlation be- tween HA1 titres and ELISA optical density (OD) values for the 3 groups of serums.

The ELISA gave excellent correlation with HA1 tests (Figure 1). Serum antibody titres to CPV from dogs in group (i) (unvaccinated, n = 13) gave very highly significant correlation, whilst titres from dogs in groups (ii) (vaccinated, CPV/D/H, n=32) and (iii) (vaccinated, D/H only, n=24) gave highly significant correlation in ELISA with HA1 (p = .05).

Only serums from CPV vaccinated dogs with high titres (HA1 > 8192) gave strong ELISA reactions with the cell antigen control (mean OD 1.88). Serums from dogs vaccinated with CPV and with lower HA1 titres gave slight reactions. Serums from dogs in the other 2 groups did not react with cell antigen (results not shown).

The ELISA was specific for antibody to CPV. Vaccinated dogs with high titres to distemper virus, serum neutralisation (SN) titre 1024, were low reactors to CPV in ELISA where CPV HA1 titre < 4; thus a generalised immune response was not being measured.

The high correlation between ELISA and HA1 for serums from vaccinated or unvaccinated dogs indicates the ELISA could be an ideal replacement for the HA1 test for antibody to CPV.

CPV vaccinated dogs often have high antibody titres to cell culture antigens. This is not unexpected given the methods of production of present day CPV vaccines. Fiscus et al (1985) described a rapid ELISA with good correlation with HA1 and SN. However this assay does not consider the likelihood of the ELISA detecting antibodies to cell culture antigens. We have further observed recognition of cell culture antigens by serums from CPV vaccinated dogs in immunoblot procedures.

Australian Veterinary Journal, Vol. 64, No. 7, July, 1987

Thus the serums of vaccinated dogs may sometimes give positive reactions in ELISA while not aquiring protective levels of antibody if the ELISA viral antigen is not sufficiently purified.

Other ELISAs for CPV antibody have been reported (Rice et al 1982; Nara et al 1983) and have found favourable prognosis was reflected by occurrence of high levels of co- proantibody. High serum titres in HA1 and ELISA were also demonstrated for these dogs, with serum CPV IgG and IgM antibody levels correlating well in both tests.

The present assay utilises a highly purified CPV antigen. The high correlations of ELISA with HA1 and no cell antigen reactions for all vaccinated dogs except for those with very high HA1 titre suggest that little, if any, cell antigen reactive with sera from vaccinated dogs exists in the ELISA antigen. Work is presently in progress to confirm this using immu- noblotting techniques.

The use of a monoclonal antibody to CPV as the initial solid-phase antigen absorbent would ensure reactions with canine serums specific for CPV antigens only. We are presently defining the antigen specificity of a haemagglutinating mon- oclonal antibody to CPV for this purpose.

It is envisaged that a second generation ELISA test would be rapid, requiring less than 1 h for results. It could be produced in kit form, enabling its use in veterinary clinics and allowing confirmation of the timing or efficacy of vaccinations for pups, or the determination of protective titres in conva- lescent or exposed dogs.

We are grateful for the continuing support from the Gabba Greyhound Club, Brisbane, Queensland.

References Carmichael L E, Joubert J C and Pollock R V H (1983) - Cornell

Fiscus S A, Mildbrand M M, Gordon J C, Teramoto Y A and

Johnson R H and Spadbrow P B (1979) - Aust Vet J 55: 151 Joo H S, Donaldson-Wood C R and Johnson R H (1976) - Aust

Nara P L, Winters K, Rice J B, Olsen R B and Krakowka, S (1983) -

Rice J B, Winters K A, Krakowa S and Olsen R G (1982) - Infect

Smith J R and Johnson R H (1986) - Vet Rec (in press)

Vet 13: 13

Winston S (1985) - A m J Ver Res 46: 859

Vet J 52: 422

A m J Vet Res 44: 1989

Immun 3 8 1003

(Accepted for publication I0 March 1987)

The morphology of the ovine placenta in pregnancy toxaemia

Department of Anatomy, University of Melbourne, Parkville, Victoria 3052

G M MITCHELL B F STRATFORD

Ovine toxaemia of pregnancy has long been associated with nutritional inadequacy, harsh climatic conditions, multiple pregnancy and overweight ewes (Seaman 1854). Neuromus- cular disturbances are a major feature of the disease (Mc- Clymont and Setchell 1955) with hypoglycaemia (McClymont and Setchell 1955) and proteinuria also reported (Ferris et al 1969). Foetal death in utero is common, although affected ewes generally recover after delivery (Assali et a1 1958).

There have been no previous reports on the uterine and placental morphology of animals with this disease, although the pathophysiology of the liver, kidneys, adrenals and nervous system has been extensively examined. In this study the pla- centa and uterine vasculature from normal and experimentally induced toxaemic ewes in the latter part of pregnancy were examined for significant morphological and morpho- metric changes.

From day 40 of pregnancy 10 Corriedale ewes had their grazing restricted to a poorly grassed paddock supplemented

22 1