Detection of antibodies to Eperythrozoon ovis by the use of an enzyme-linked immunosorbent assay

Research in Veterinary Science 1987, 43, 249-252

Detection of antibodies to Eperythrozoon ovis by the use of an enzyme-linked immunosorbent assay F. M. LANG, G. R. FERRIER, T. J. NICHOLLS, Victorian Department of Agriculture and Rural Affairs, Regional Veterinary Laboratory, PO Box 388, Benalla, 3672 Australia

Specific antibody to Eperythrozoon ovis was detected by an enzyme-linked immunosorbent assay (ELISA) in the sera of infected sheep. In the presence of parasite antigen, positive control serum showed a reaction approximately eight times that of negative serum. When compared to an immunofluorescent antibody test (I.-AT), the ELISA was eight times more sensitive. Positive control sera gave a titre of 1:3200 by IFAT and 1:25,600 by ELISA. Through the use of a reference titration curve ELISA could be used as a semi-quantitative system to determine antibody levels in test sera.

EPERYTHROZOON ovis is a rickettsia-like parasiIe which is found associated with the outer surface of the ovine erythrocyte (Neitz et al 1934, McKee et al 1973). Parasitaemia is ofIen but not always associated with anaemia, ill thrifI and occasionally death (Valli 1985). Infection of sheep with E ovis has been reported in Africa, the USA, Australia, England, New Zealand and Scandinavia (Neitz et al 1934, Littlejohns 1960, Foggie 1961, Jolly 1967, Overas 1969, McKee et al 1973, Ilemobade and Blotkamp 1977). Diagnosis of infection is usually based on the presence of the organisms in Giemsa stained blood smears. Campbell et a1 (1971) considered that establishing a· diagnosis of active E ovis infection is difficult, because the parasitaemia is transient and sheep do not usually show parasites in blood smears by the time clinical signs are apparent. Alternative means of diagnosis, based on the detection of antibodies to E ovis, have been described. These include a complement fixation test (CFT) (Daddow 1977), immunofluorescent antibody tests (IFAT) (I1emobade and Blotkamp 1978, Maughan 1981, Nicholls and Veale 1986) and a modified anti-globulin (Coombs) test (Sheriff and Geering 1969). As part of investigations to improve an IFAT, the fortuitous separation of E o vis from erythrocytes was achieved (Lang et al 1986). The present study was subsequently undertaken to determine if separated organisms were suitable as antigen for use in an enzyme-linked immunosorbent assay (ELISA) specific for this parasite. To the authors' knowledge this is the

first report of an ELISA specific for detecting antibodies to E ovis. The aims of the work reported here were, first, to establish the parameters for carrying out an ELISA suitable for detecting antibodies to E ovis and, secondly, to assess the sensitivity and methodology of the ELISA relative to a conventional IFA T.

Materials and methods

Animals and infection Serum and whole blood used in this study originated from Merino cross or Corriedale sheep maintained under insect-free conditions. The animals were experimentally infected by the single intravenous inoculation of approximately 108 parasitised erythrocytes which were originally obtained from a naturally infected sheep (Nicholls and Veale 1986).

Determination of the degree of infection Venous blood was collected into evacuated tubes containing ethylenediaminetetra acetic acid (BectonDickson). The degree of infection was expressed as a percentage of parasitised cells afIer a count of 400 erythrocytes (Ilemobade and Blotkamp 1978).

Separation of parasites Peripheral blood, with greater than 90 per cent infected etythrocytes, was collected from two sheep (EI and E2) and parasites were separated as described by Lang et al (1986). Briefly, parasitised erythrocytes were washed in buffered saline before being lysed in a hypotonic solution. Parasites were then obtained by differential centrifugation. The parasite preparation contained 11· 5 mg ml I of protein and was used without further treatment as antigen in an ELISA. Peripheral blood from an uninfected sheep, E2 before infection, was similarly processed to obtain a parasite-free lysed cell preparation.

249

250

F. M. Lang, G. R. Ferrier, T. J. Nicholls

Serum samples Positive control serum was obtained from sheep E308 10 days after the peak of infection (>90 per cent). Negative control sera were obtained from sheep EI and E2 before infection with E ovis. Sera from routine submissions to this laboratory were also tested by ELISA. ELISA

Optimal antigen and conjugate dilutions were established by checker-board titrations using control sera from uninfected and infected sheep. Antigen was diluted in 50 mM sodium carbonate buffer pH 9·2 and 100 IJI volumes were added to wells of a polystyrene microtitre plate (Cooke type M 24A) and incubated at 37°C for 90 minutes or at 4°C overnight. Plates were then washed with phosphate buffered saline (PBS, 10 mM phosphate, 150 mM sodium chloride, pH 7· 2) containing O' 05 per cent Tween 20 (PBS-Tween). Serum samples, diluted in PBs-Tween were added in duplicate to the wells (100 IJI per well). Following incubation at 37°C for 90 minutes, the plates were again washed with PBs-Tween and 100 IJI of affinity purified rabbit anti-ovine IgG conjugated to horseradish peroxidase (by the method of Wilson and Nakane 1978) was added to each well. Plates were incubated at 37°C for 90 minutes and then washed before the addition of peroxidase substrate consisting of I mM 2,2' azino-dilethylbenzthiazolin-sulphonate (6)] and 2· 5 mM hydrogen peroxide in 100 mM citrate/phosphate buffer, pH 4·2. The optical density at 414 nm (OD 414) was read, 30 minutes after addition of 100 IJI of substrate, on a Titertek Multiskan Me Plate Reader (Flow). Unhydrolysed substrate was used to set the instrument to zero.

Zeiss incident light fluorescence microscope using an excitation waveband of 450 to 490 nm. Results

Antigen and conjugate dilutions Checker-board tit rations with control sera from uninfected and infected sheep showed the optimal antigen dilution to be I:500, equivalent to 22' 4 IJg protein ml- I (2' 2 IJg protein per well). Reactions by control sera to parasite antigen preparations from sheep EI and E2 showed no significant differences and for convenience parasite antigen from sheep EI was used in subsequent experiments. The conjugate was found to be optimally diluted at 1:20,000.

Specificity of antibodies for E ovis and positive/ negative differentiation In ELISA, sera from infected and uninfected sheep showed no reaction (OD 414<0'10) to parasite-free material prepared from blood from uninfected sheep E2. Data in Fig 1 show the effect of increasing serum dilution on the OD 414 for positive control serum (sheep E308). Dilutions of negative sera greater than 1:200 showed no marked decrease of OD 414 values below 0'10 (results not shown). A negative reference point estimated as the mean OD 414 ± 2 SD of 20 replicates of a 1:200 dilution of serum from an uninfected sheep, was 0·10 ± 0'09. The positive control serum when tested at a 1:200 dilution differed by approximately 0·80 OD 414 units from the reference negative point (Fig 1). The ratio of OD 414 values for positive and negative sera under these conditions was 8: I. 1·0

Specificity of antibodies for E ovis Specificity of antibodies for E ovis was tested by using blood from uninfected sheep E2 as a source of parasite-free coating material in ELISA. A negative reference point was established by determination of the mean OD414 ± 2 SD of 20 replicates of serum from an uninfected sheep.

Indirect fluorescent antibody test Microscope slides coated with parasitised erythrocytes were prepared and used as described by Nicholls and Veale (1986). Antibodies in positive and negative control sera were detected with a 1:20 dilution of fluorescent labelled rabbit anti-sheep immunoglobulin (Wellcome Laboratories). Washing stages were in PBS and slides were examined with a

0·9

E

0'8

'" :!

0·7 0·6

.~

0·5

E

>-

c

'" 'a0 '0

'iii o

G.'iEIJ 0

l''')

Before E avis infection After E avis infection (

I"'l ('0)

(

0·4

..

(

..

...

)

)

)

1'1

0·3

I-I

I-I

0·2 0·1 0

2

4

8 Sheep E1 Sheep E2

FIG 1: Comparison of detecting antibody to Eperythrozoon avis using ELISA (optical density at 414 nml and IFAT (results in bracketsl. Serum samples from uninfected and infected sheep.••• Strong fluorescent labelling. •• Moderate fluorescent labelling. • Weak fluorescent labelling, - No fluorescent labelling

ELISA

for antibodies to Eperythrozoon ovis

Comparison of ELISA and IFA T By ELISA, positive control serum (sheep E308) could be titrated to a dilution of I :25,600 in the ELISA and to 1:3,200 in the IFAT (Fig I). This is an eightfold increase in sensitivity of the ELISA compared to [FAT.

Measurement of antibody by the use of a reference titration curve Sera from sheep EI and sheep E2 collected 10 days after peak infection were tested at a single dilution point (I :200) in the ELISA and compared to a titration curve of the positive control serum as a reference (De Savigny and Voller 1980). The OD 414 values for the sera of sheep EI and sheep E2 at a single dilution point were 0·60 and 0'80, equivalent to dilutions of I:800 and I :400 of the positive reference serum. Both sera were positive by IFAT up to a I :200 dilution. Other ovine sera (n = 20) from routine submissions to this laboratory tested at a single dilution point showed OD 414 values less than 0'40, equivalent to dilutions greater than I :3200 of the positive reference serum. Discussion The results demonstrate that an ELISA can be developed capable of detecting antibodies to E avis. Since only a small number of sheep were used as part of this study, further evaluation studies in a large number of sheep must be performed to determine the absolute specificity of the assay. The presence of erythrocyte membrane material in the parasite antigen preparation (Lang et al 1986) may result in naturally occurring blood group antibodies in some sheep sera (Tucker 1975) contributing to OD 414 values. Nonspecific reaction was not found in the present study and results from other studies where CFT (Daddow 1977), antiglobulin tests (Sheriff and Geering 1969) and IFAT (llemobade and Blotkamp 1978, Nicholls and Veale 1986) were used indicate that false positive reactions should be unlikely. When compared to the IFAT the ELISA showed an eightfold increase in sensitivity. While the IFAT is specific and relatively easy to evaluate, there are contradictory reports of low and high antibody titres in response to infection. Hung and Lloyd (1985) and lIemobade and Blotkamp (1978) reported IFAT antibody tit res of 1:4 to I :640, while Nicholls and Veale (1986) found titres of I :3000 being maintained in experimentally infected sheep. The [FAT is subjective and needs strict control procedures to achieve reproducible estimates of antibody titres. Furthermore, the IFAT is not amenable to large throughput of samples hence limiting its use as a diagnostic tool on a flock basis. In addition to increased sensitivity, the oppor-

251

tunity for the use of standardised antigen and control serum preparations in ELISA will overcome the shortcomings of IFAT and enable less subjective serological analyses to be undertaken. Daddow (1977) used a frozen-thawed, sonicated extract of infected erythrocytes as antigen for a CFT and showed that sera from infected sheep generally gave complement fixing tit res less than I :64. The CFT appears, a priori, to lack high sensitivity and in addition, some sheep gave detectable tit res for only a short time or even failed to do so. The ELISA described in this paper exhibited high sensitivity and overcame potential problems associated with the CFT, such as samples with anticomplementary activity or little or no complement fixing antibody. The range of OD 414 values found when a small number of routine submissions to this laboratory were tested suggested a persistence of low antibody levels to E avis. lIemobade and Blotkamp (1978) reported that antibody responses may persist for up to two years and may be related to possible E avis strain differences. Inability to diagnose infection accurately and reports of various levels of virulence of E avis (Sheriff et al 1966, Overas 1969, Campbell et al 1971) has lead to eperythrozoonosis remaining a disease of ill-defined economic significance in the sheep industry. Extended work using ELISA offers efficient and sensitive serological testing which should help determine whether the persistence and magnitude of antibody responses can be related to infection status and, or, E avis strain difference. Because of the advantages discussed above, the ELISA for E avis should prove to be a valuable aid in experimental and epidemiological studies and differential diagnosis. In addition quantitative studies of antibody responses could allow discrimination between clinical and subclinical infections and their contribution to the complex interacting effects of nutrition, management and helminth burdens. Furthermore, the principles of the method may also have application to similar blood parasites such as E suis and Haemobartonella felis. Acknowledgements We wish to thank staff members of the Regional Veterinary Laboratory for their helpful discussions and Ms R. Hussell for the preparation of the manuscript. F.M.L. undertook this work as part fulfilment of BAgSc (Hons) degree at LaTrobe University, Melbourne, Australia. References CAMPBELL, R., SLOAN, C. & HARBUTT, P, (1971) Australian Veterinary Journal 47, 538-541 DADDOW, K, (1977) Australian Veterinary JournalS3, 139-143

252

F. M. Lang, G. R. Ferrier, T. J. Nicholls

DE SAVIGNY, D. & VOLLER, A. (1980) Journal of Immunoassay I, 105-128 FOGGIE, A. (1961) Veterinary Record 73,453-454 HUNG, A. & LLOYD, S. (1985) Research in Veterinary Science 39,275-278 ILEMOBADE, A. & BLOTKAMP, C. (1977) Veterinary Record 101, 153-154 ILEMOBADE, A. & BLOTKAMP, C. (1978) Zeitschrift fur Tropenmedizin und Parasitologie 29, 307-310 JOLLY, R. (1967) New Zealand Veterinary Journal 15, 47-48 LANG, F., FERRIER, G. & NICHOLLS, T. (1986) Veterinary Record 119, 359 LITTLEJOHNS, I. (1960) Australian Veterinary Journal 36, 260-265 MAUGHAN, J. (1981) MSc thesis, La Trobe University McKEE, A., ZIEGLER, R. & GILES, R. (1973) American Journal of Veterinary Research 34, 1196-1201 NEITZ, W., ALEXANDER, R. & DU TOIT, P. (1934) Onderstepoort Journal of Veterinary Science and Animal Industry 3, 263-271

NICHOLLS, T. & VEALE, P. (1986) Australian Veterinary Journal 63,157-159 OVERAS, J. (1969) Acta Veterinaria Scandinavica Supplement 28, 1-148 SHERIFF, D., CLAPP, K. & REID, M. (1966) Australian Veterinary Journal 42, 169-176 SHERIFF, D. & GEERING, M. (1969) Australian Veterinary Journal 45, 505-507 TUCKER, E. (1975) The Blood of Sheep. Ed M. Blunt. Berlin, Springer-Verlag. p 134 VALLI, V. (1985) Pathology of Domestic Animals, Volume 3, 3rd edn, Eds K. Jubb, P. Kennedy and N. Palmer. New York, Academic Press. p 160 WILSON, M. & NAKANE, P. (1978) Immunofluorescence and Related Staining Techniques. Eds W. Knapp, K. Holibar and G. Wick. Amsterdam, Elsevier. pp 215-224

Received July 18, 1986 Accepted August 15, 1986