B,. vel. J. (1990). 146, 364

USE OF THE IDEIA* ELISA TO DETECT CHLAMYDIAPSIITACI (OVIS) IN MATERIAL FROM ABORTED FETAL

MEMBRANES AND MILK FROM EWES AFFECTED BYOVINE ENZOOTIC ABORTION

ROSE TIIOMASt, HELEN C. DAVISON; and A.]. WILSMORElI

Department of Velninary Medicine andAnimalHusbandry, Royal Velerinary College, BoltonsPark, Potters Bar, Hms. EN6 THB

SUMMARY

The IDEIA ELISA was used to detect Chlamydia psittaci (avis) antigen in ewes'milk to which were added serial dilutions of chlamydiae titrated as inclusionforming units (ifus) in McCoy cell tissue culture. The test was able to detect asfew as 35 ifus/ml of the organism.

The ELISA was then used to detect chlamydial antigen in fetal membranesand milk from ewes clinically affected with ovine enzootic abortion (OEA). Theresults were compared with results of isolation of chlamydiae in McCoy celltissue culture from the same material. The fetal membranes of t 7 of t 9 eweswere positive for chlamydia when tested with the ELISA but chlamydia could becultured from only t 5 of them. Milk samples from 26 ewes which had abortedbetween t and 34 days previously were tested: chlamydiae could not be culturedfrom any of them and only one was positive when tested by the ELISA. Theresults show that the IDEIA ELISA is a sensitive test for the detection of C.psittaci (avis) antigens. The positive results to this test for; the three samples fromwhich chlamydiae could not be cultured suggest that the test is not as specific asculture or that it detected dead organisms. Chlamydiae do not appear to beexcreted in the milk of ewes affected with OEA.

INTRODUCTION

Traditionally, the detection of Chlamydia psittaci (avis), the organism responsible for ovineenzootic abortion (OEA), has been achieved by isolating it in fertile hens' eggs and intissue culture. Both these methods are time consuming and depend on the presence ofviable organisms in the sample to be tested. Wills et al. (1986) used the IDEIA chlamydia

·IDEIA, trade name for 'an amplified enzyme-linked immunoassay for the detection of Chlamydia in humanclinical specimens'.

tPresent address: CTVM, University of Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG.*Present address: Regency House, Bow Street, Langport, Somerset.'Correspondence to A.J. Wilsmore.

ELISA FOR DIAGNOSING OVINE ENZOOTIC ABORTION 365

test (Novo Biolab, St. Johns Innovation Centre, Cowley Road, Cambridge, CB4 4WS),an amplified ELISA for the detection of Chlamydia in human clinical specimens whichtakes approximately 6 h to perform, to detect C. psittaci antigen in conjunctival scrapesfrom cats. Souriau & Rodolakis (1986) used an ELISA to detect chlamydial antigen inswabs from aborted ewes and goats . In this experiment, the IDEIA ELISA was used todetect the presence of C. psittaci (ovis) in aborted ovine fetal membranes and milk fromewes affected by OEA. The organism is a potential zoonosis and has been identified as acause of abortion in pregnant women who have had contact with affected sheep (Buxton,1986). Sheep dairy products are becoming more popular and it is important to determinewhether milk from affected animals is a source of infection for man or lambs.

MATERIALS AND METHODS

In order to demonstrate that the IDEIA chlamydial antigen detection system detected C.psittaci (ovis) antigen, the organism was first grown in the yolk sacs of embryonated eggs.The infected yolk sacs were titrated in McCoy cell culture and the chlamydiae presentwere quantified as inclusion forming units (ifus). A suspension containing 1120 ifus wasprepared in phosphate-buffered saline and then diluted in milk from an uninfected eweto give the following dilutions: 560,280, 140, 70, 35 and 18 ifus/ml. Two hundred ,ul ofeach dilution was tested with the IDEIA ELISA and the absorbance value of the result ofeach test was read at 492 nm using an MR 600 photometer (Dynatech LaboratoriesLimited, Daux Road, Billinghurst, West Sussex).

To demonstrate chlamydial antigen in the fetal membranes of aborted sheep, swabswere taken from the fetal membranes of 19 ewes in which chlamydial infection hadalready been demonstrated in 15 of the membranes by microscopical examination andby culture. Each swab was placed in 1 ml of the transport medium supplied with theIDEIA test kit and agitated for 15 s using a whirlimix. They were then stored at -70·Cuntil the test was performed when they were thawed and re-agitated. The swabs werethen discarded and the transport medium remaining was tested by the IDEIA methodand the absorbance value for each test was read as above.

To investigate the presence of chlamydiae in the milk of ewes which had experiencedovine enzootic abortion (OEA), milk samples were obtained from 26 ewes which hadaborted between 1 and 34 days previously, and prepared for cultural examination forchlamydia and testing for chlamydial antigen using the IDEIA test kit.

Each milk sample was centrifuged at 4000 g for 1 h. The fatty layer and supernatantwere discarded and the remaining cellular fraction was mixed in 1 ml of the IDEIAtransport medium and used for the test.

RESULTS

Figure 1 shows the results of applying the IDEIA ELISA to the serial dilutions of thesuspension of chlamydiae. The test was able to detect chlamydial antigen in sampleswith as little as 35 ifus/ml.

Chlamydial antigen was detected in 17 of the 19 samples from fetal membranesincluding the 15 which had been detected by microscopical examination and culture.

366 BRITISH VETERINARY JOURNAL. 146, 4

2.0•

1.5

0 .5

•• •o _~~I_O!! J~_l?~~L • .

-0.1 35 18 01

Fig. 1. Absorbance values of serial dilutions of viable chlamydiae tested by the IDEIA ELISA.

Table IResults of the IDEIA test and of cultural examination of milk samples from aborting

ewes

Number of Numberof etues Results of Results of celldays after sampled /DEJA ELISA cultur«abortion

1 4 -ve -ve2 1 - ve -ve3 7 - ve -ve5 3 - ve -rve6 3 -ve -we7 1 -ve -ve8 1 - ve -ve

11 1 - ve - ve13 1 +ve -ve14 1 -rve -ve18 1 -ve -ve26 1 -rve -ve34 1 -ve -ve

ELISA FOR DIAGNOSING OVINE ENZOOTIC ABORTION 367

The test detected chlamydial antigen in two other samples in which chlamydiae had notbeen demonstrated by microscopical examination or culture.

Table I shows the result of the IDEIA ELISA and culture of the milk samples fromaborting ewes. Only one milk sample taken from a ewe 13 days after it had aborted waspositive to the IDEIA ELISA. However, chlamydiae could not be cultivated in McCoycell tissue culture which had been inoculated with the milk.

DISCUSSION

The ability of the IDEIA ELISA to detect chlamydial antigen in samples that containedas little as 35 ifus of C. psittaci (ovis) per ml indicated that the test was vel)' sensitive, moreso than described previously where it was shown to detect C. psittaci antigen in conjunc­tival scrapes from cats up to a dilution equivalent to 157 ifus per ml (Wills et al., 1986).

The sensitivity of the test was further demonstrated when it gave a positive result for 17of 19 samples from aborting ewes. However, the diagnosis of OEA was supported byculture and microscopical examination in only 15 of the cases. This may mean that thetest was less specific than culture or that chlamydial antigen was present but not in theform of viable organisms. A more extensive evaluation of the test might indicate if theformer was the case: for example, other bacterial lipopolysaccharide antigens, which maybe a source of cross-reaction, could be assayed or placental swabs from hill flockswith norecorded disease could be tested.

When milk samples taken from aborting ewes were tested, chlamydial antigen couldbe detected in only one of 26 samples by the IDEIA ELISA and in none of them byculture. This also may indicate either a lack of specificity or that chlamydial antigen inthe milk sample was not present as viable organisms. The results of this experiment showthat chlamydiae are not likely to be excreted in the milk of infected ewes. This supportssimilar observations made by Jones & Anderson (1989) and Venables et al: (1989).However, there is a danger of the milk becoming contaminated with chlamydiae fromvaginal discharges of affected ewes which can run down the back of the udder on to theteats.

REFERENCES

BUXTON, D. (1986). Vtttrinary Rtcord 118,510.JONES, G. E. & ANDERSON, I. E. (1989). Vtttrinary Record 124,562.SoURIAU, A. & RODOl.AKlS, A. (1986). Vtttrinary Microbiology 11, 251.VENABLES, C., DAWSON, M. & BASKERVILLE, M. (1989). Vtttrinary Record 124, 137.WILLS,]. M., MILLARD, W. G. & HOWARD, P. E. (1986). Vtttrinary Rtcord119, 418.

(Accepted forpublication 9 October 1989)