1974) to selectively increase the numbers of C. fetus prior to culture on solid selective medium. In this letter we describe the use of this enrichment medium to transport and culture samples of preputial liquid. The medium is prepared by adding 300 pg per rnl of 5-fluorouracil, 100 units per mI of polymyxin B sulphate, 50 ,ug per ml of brilliant green, 3 ,ug per ml of nalidixic acid and 100 pg per ml of cycloheximide to ox serum which is then dispensed in 10 ml volumes into screw-capped 28 ml universal containers. These containers should be new with well-fitting metal lids and rubber wads. A small hole, 1 mm in diameter, is drilled through the centre of the metal lid, but not through the rubber wad. The containers are next put into a waterbath which is then brought to boiling. The solidified medium is then broken up with a sterile glass rod or pipette, and air in contact replaced with a gaseous mixture composed of 5 % oxygen, 5% carbon dioxide and 90% nitrogen. The prodecure is to loosen the caps, put containers in a Mclntosh and Fildes jar, partially evacuate the jar to 19 cm mercury, introduce carbon dioxide until the internal pressure reaches 23 cm mercury and then nitrogen until the internal pressure reaches atmospheric pressure. Next, remove the containers carefully from the jar, screw caps down tightly and store in a refrigerator for approximately 1 week prior to use. This period of cold storage, in which the medium becomes deep green, makes it more selective against organisms such as Spirillum spp. Stored in a refrigerator, the prepared containers of medium remain suitable for use for at least 3 months. Preputial liquids are collected by plastic preputial pipette (Bartlett er a / 1947) and washed into 4 ml of sterile physiological saline. This sample is stood briefly to allow epithelial cells to settle, and then 1 ml of supernatant liquid withdrawn using a sterile syringe and needle and injected into the universal container through the small hole in the centre of the metal lid. The 1 ml sample is mixed into the medium by shaking the container. During inoculation, it is important to avoid injecting air or removing the lid of the universat container. The inoculated containers should be wrapped in paper within insulated boxes and forwarded so as to be received at the laboratory within 2 days. The most suitable temperature range during transport is 18°C to 37°C and care should be taken to avoid low temperature (O°C to 5°C).

After arrival at the laboratory, the inoculated container is incubated at 37°C for 4 days. Two or 3 ml of sterile physiological saline is added, mixed in thoroughly and then all available liquid removed with a 10 rnl pipette and delivered into a sterile tube. This liquid is then examined for the presence of C. fetus subsp. fetus by culture and immunofluorescence. For culture, liquid is filtered through an A P 2501300 13 mm diameter prefilter and a 0.65 pn Millipore DAWP 01300 13 rnm diameter filter using a syringe and filter holder and then 0.1 ml per plate distributed over the total surface of 1 or 2 plates of solid selective medium using a bacteriological swab. The laboratory procedures for the incubation, cultural isolation and identification of C. fetus subsp. fetus and the methods for demonstration of the organisms by immunofluorescence are described in the paper by Dufty (1967). This method has been used on numerous occasions since 1974 to demonstrate C. fetus in samples collected from bulls in the field. In a trial in which 89 separate samples were collected from 5 infected bulls and transported and cultured in the medium, C. feetus subsp. fetus was demonstrated in 84 (94.4%) by immunofluorescence and 77 (86.5%) by culture. B. L. CLARK, J. H. DUFTY, CSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, P.O., Parkville, Victoria, 3052 20 January 1978

References Bartlett, D. E., Hasson, Eleanor V. and (1947)-J. Am. vet. med. Ass. 110: 114. Clark, B. L., Monsbourgh, Mary J. and (19741-Ausr. vet. J . 50: 324. Dufty, J . H . (1967)-Aust. vet. J . 43: 433. Mellick, P. W . , Winter, A. J. and ( 1965)- Cornell Vet. 55: 280. Shepler, V. M., Plumer, G . J. and Faber, J. E. vet. Res. 24: 749.

Teeter, K. G . Dufty, J . H. McEntee,

K.

(1963)-Am.

J.

ANTIGENIC HOMOGENEITY OF EQUINE ADENOVIRUSES

Equine adenoviruses (EAdV) have been isolated from conventional foals without and with respiratory disease and from Arabian or part Arabian foals with primary, severe, combined immunodeficiency disease (PSCID). Evidence has been presented that some of these EAdV isolates are antigenically closely related (Studdert et a1 1974; Thompson et a1 1976). The isolation of 4 additional EAdV strains from 6 PSCID foals (material apropriate for virus isolation was not available from two of the foals) in the 1976/77 foaling season provided an opportunity to make additional antigenic comparisons between Australian EAdV strains. The 4 EAdV strains designated EAdV -M2, -M3, -M4 and -M5 were isolated from either nasal swabs antemorem or from lung tissue collected postmortem by inoculation of equine foetal kidnev (EFK) cell cultures using procedures previously described (Wilks and Studdert 1973). Stocks of each virus were prepared after 5 passages in EFK cells. The 4 viruses were compared to each other and to our standard strain EAdV-MI ( W i l k s a n d S t u d d e r t 1973) in r e c i p r o c a l - c r o s s haemagglutination-inhibition (HI) and serum neutralisation (SN) tests performed as previously described (Studdert et al Australian Veterinary Journal, Vol. 54, May, 1978

1974). Antiserums to the 4 newly isolated EAdV were raised in rabbits. Rabbit antiserums t o EAdV-MI, EAdV-Q (Harden el al 1972) and EAdV-67 (Thompson et a1 1976) and to EAdV-Col (a strain isolated from a PSCID toal in Colorado; A. L. McChesney personal communication, were also included in the antigenicanalyses. Serum from a mare(Bithynia)collected 6days after her 1973 foal died of PSCID (foal number 17 in the report of Thompson et a/ 1975) which was also the foal from which EAdV 67 (Thompson et a / 1976) was isolated, was also included in the tests. By H I the degree of antigenic homogeneity among the 5 EAdV strains was striking. Most H I titres of a particular serum were identical; exceptions were within 1 double dilution. By SN (Table 1) there was again a high degree of antigenic homogeneity although some exceptions appear. For example EAdVM4 antiserum had a titre of 3043 against homologous virus but only 95 against EAdV-M5. It might be noted, however, that the amount of EAdV-M5 virus in the test is a little greater than 4 times that in the homologous reaction. In fact the data presented are representative of the best balanced data obtained in a single experiment. Other repeats of the assays (data not

263

TABLE 1 Comparison of 4 Equine Adenoviruses by Serum Neutralisation Tests Antibody

Virus

CrCID50)

M1 (152) M2 (90) M3 (1 80) M4 (76) M5 (3 16)

MI =6*

Q

67

M2

M3

M4

M5

Col

1279

320

1280

3043

538

Bithynia

3619

760

1522

1810

3043

2735

3043

1810

1810

NT

6086

452

905

6354

905

1810

m 3

5 38

NT

8607

380

3043

1810

7258

5118

1810

3043

NT

14474

380

452

1280

380

540

1520

3620

1076

320

95

* Reciprocal of highest serum dilution neutralising the stated amount of virus. Homologous titre is underlined. NT = not tested. shown) underlined the observation that there is a high degree of antigenic homogeneity among EAdV. The 4 PSCID foals from which EAdV were isolated came from 3 geographically well-separated areas in Victoria and NSW. These data together with earlier data (Studdert et a1 1974; Thompson et a/ 1976) support the contention that EAdV are at present represented by a single antigenic type, a fact favourable to the control of EAdV disease by vaccination. It is somewhat unusual to find such a high degree of antigenic homogeneity within adenovirus isolates from a single mammalian species. In man 31 antigenic types are recognised and comparable figures for other species include simian 12, bovine 3, porcine 3, dog 2. We are grateful to the Australian Equine Research Foundation for financial support. M. J. STUDDERT,

264

School of Veterinary Science, University of Melbourne, Parkville, Victoria 3052 12 January 1978

References Harden, T. J . , Pascoe, R. K. and Spradbrow, P. B. (1972)-Aust. vet. J. 48: 478. Studdert, M. J., Wilks, C . R. and Coggins, L. (1974)-Am. J. vet. Res. 35: 693. Thompson, D. B., Spradbrow, P. B. and Studdert, M. J. (1976)-Aust. vet. J. 52: 435. Thompson, D. B., Studdert, M. J., Beilharz, R. G. and Littlejohns, I. R. (197S)-Aust. vet. J. 51: 109. Wilks, C . R. and Studdert, M. J. (1973)-Aust. vet. J. 49: 456.

Australian Veterinary Journal, Vol. 54, May, 1978

Antigenic homogeneity of equine adenoviruses.

1974) to selectively increase the numbers of C. fetus prior to culture on solid selective medium. In this letter we describe the use of this enrichmen...
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