A microplate enzyme immunoassay for detecting and measuring an tibodies to babesia bovis in cattle serum

D. N. BARRY*, B. J . RODWELLt, P. TIMMS* and W. McGREGOR*

SUMMARY A microplate enzyme immunoassay (EIA) is described for measuring IgG antibody to 6abeSh bovis in cattle serum. 6. 60vis antibody status (whether positive or negative) and the amount of 6. 60vis antibody (EIA score), were measured by comparison with reference serums. The EIA was shown to be specific for 6. Bovis, and EIA score correlated well with EIA titre.

Comparison of EIA wlth the indirect Fluorescent Antibody Test (IFAT) showed more than 95% agreement between the methods and disagreement in only 1.6% of serum samples tested. The remaining 3.2% were positive by EIA and suspected positive by IFAT. The EIA was shown, by titrating positive serums, to be more sensitive than IFAT, which explained its tendency to detect more positive serums than IFAT.

EIA detected B. bovis antibody in experimentally infected cattle by day 14 post infection (pi) and for at least 268 days pi. EIA score for 6. bovis antibody in immune cattle Increased significantly (~(0.05) following heterologous strain challenge. Aust. vet. J . 59:

Introduction Highly infective blood-based vaccines of Babesia bovis,

Babesia bigemina or Anaplasma centrale parasites have been produced and distributed from this laboratory since 1964 (Callow 1977). One requirement for vaccine production is that calves to be used as donors of infected blood be free of antibodies to B. bovis, B. bigemina and A . centrale or A . marginale. To ensure this, calves are obtained from a tick- free area of south east Queensland and routinely screened by indirect fluorescent antibody test (IFAT). This test, while very useful, has several disadvantages in that subjective judgment is required in reading it (Callow et a1 1974) and it has proved less than satisfactory in this laboratory for B. bigemina and the anaplasms. Enzyme immunoassay (EIA) methods, which are more objective than the IFAT, have been reported for measuring antibodies to B. divergens (Purnell et a1 1976) and A . marginale (Thoen et a1 1980). We have, therefore, begun evaluating EIA methods to measure anti- bodies to B. bovis. B. bigemina and A . marginale and report here an EIA method, standardised by reference serums, for detecting and measuring immunoglobulin G (IgG) antibody to B. bovis in cattle serum, with comparisons between the EIA and IFAT.

Materials and Methods Antigen Preparation

Blood, with a parasitaemia of approximately 20%, was collected into preservative-free heparin, from a splenectom- ised calf infected with K strain B. bovis. Parasitised eryth- rocytes were concentrated by a modification of the method of Mahoney (1967) using 0.425% potassium chloride instead of sodium chloride. Packed cells were diluted 4-fold with phosphate buffered saline (PBS) and leucocytes removed by mixing with an equal volume of C F l l cellulose in PBS (0.17 g/ml) and standing for 30 min at 4°C with gentle stirring. Parasitised erythrocytes were separated from cellulose by filtration through cotton wool, and then lysed by the method of Dodge et a1 (1963) to remove haemoglobin. The hamo- globin-free, erythrocyte ghosts were suspended in 2 volumes

of PBS, frozen and thawed, then ultrasonicated for 3 periods of 20 sec with a Biosonik 1V* using the 3/8 in (9.525 mm) probe, low power mode, at 75% power. Sonicated material was centrifuged at 23,000 g (Sorvall RC2, SS34 head) for 30 min. The resulting supernatant was used as the antigen (protein concentration 2.0 mg/ml by Biorad Protein Esti- mation Kit).

Conjugate Preparation Antibovine IgG - alkaline phosphatase conjugate was

prepared by the method of Engvall and Perlmann (1972) using rabbit anti-bovine IgGt and calf intestine alkaline phosphatase (E.C. 3.1.3.1), 12U/mg protein*. Conjugate was stored at 4°C and used for 7 months a t a dilution of 1/500 and thereafter a t a dilution of 1/250.

Micro-EIA Procedure The micro-EIA method used was similar to that used by

Purnell et a1 (1976) for B. divergens. Antigen binding: B. bovis antigen was diluted 1/400 with antigen coating buffer (Voller eta1 1976) and 0.2 ml added to each 0.25 ml capacity, round bottom, microtitration tray wellt incubated for 16 h at 4°C. Serum binding: microplate wells were then given 3 x 3 min washes with washing and diluting solution (WDS) containing 0.9% w/v NaCI, 0.02% w/v NaN, and 0.05% tween 20. Serum was diluted 1/100 with WDS, and an 0.2 ml volume was added per well and incubated for 2 h at 37’C. Conjugate binding: following 3 x 3 rnin washes of the microplate wells with WDS, 0.2 ml of anti-IgG-alkaline phosphatase conjugate, diluted 1/500 or 1/250 with WDS, was added per well and incubated at 25’C overnight. Enzyme assay: following 3 x 3 min washes of the microplate wells with WDS, 0.2 ml of p-nitrophenyl phosphate ( I mg/ml in pH 9.6 carbonate buffer with lO-’M magnesium chloride) was added per well and incubated for 1 h .a t 37°C. To stop reaction, 0.02 ml per well of 1.0 N sodium hydroxide was added and, after diluting well contents 5 times with distilled water, the absorbence of p-nitrophenol released was measured at 400 nm with Varian model 634 spectro-photometer.

Queensland Department of Primary Industries, Tick Fever Research Centre, Wacol, Queensland 4076

t Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly, Queensland 4105

136

VWR Scientific Co, United States of America Miles Laboratories A u s t Pty Ltd, Mulgrave, Victoria Sigma Chemical Go, United States of America Linbro Div Flow Laboratories, Connecticut, United States of America

Australian Veferinary Journal, Vol. 59, November, 1982

Indirect Flourescent Antibody Test (IFAT) The IFAT method and derivation of the IFAT score on

a scale from 1 + to a maximum of 4t were described previously (Callow et a1 1974). Serums were tested at 1/30 dilution.

Reference Serums and Calculation of EIA Score EIA absorbance values were found to vary from day to

day and between microplates due to unrecognised variations in material or methods (compare values for reference serums in Tables 2, 4 and 5) . Therefore 2 reference serums were replicated at least 4 times on each microplate. One reference serum, designated 148, was obtained some years ago from a B. bovis carrier and gave the lowest IFAT reaction observed for a positive serum. Stored frozen in pelleted form, i t provided a standard weak positive control serum for the IFAT. In the EIA this serum also consistently gave a low absorbance value. The other reference serum, designated 6721, was from another B. bovis carrier and gave a much higher absorbance value.

Comparison of absorbance values for test serums with those of the “weak” and “strong” reference serums allowed antibody levels to be quantified without recourse to titration. To simplify expression of results, absorbance values were converted to EIA scores using the following scale: absorbance values for “weak” reference serum 148 = 1; for “strong”

reference serum 6721 = 6; difference in absorbance between reference serums divided by 5 = increment in absorbance equivalent to a unit of EIA score. Serums with EIA score of one or more were, by definition, positive.

To conserve the stock of “weak” positive reference serum as the work progressed, other positive serums were titrated to give an identical absorbance value and used as “weak” positive reference serums.

Experimental Cattle Cattle used for experimental infection were mixed breed

Bos taurus steers, obtained from a babesiosis-free area of Queensland unless otherwise stated and tested by IFAT for B. bovis serum antibody before use.

B. bovis Strains

R strains were used. Three different isolates of B. bovis, designated K, U and

Statistical Analyses Significant differences among data in Table 5 were analysed

by balanced factorial analysis of variance (Snedecor and Cochran 1967). The best fit line and correlation analysis for the data in Figure 3 was calculated by linear regression analysis (Snedecor and Cochran 1967).

TABLE 1

Effect of serial dilution of 6. bovis antigen on EIA’ antibody score of 4 graded antiserums diluted 1/100

Animal No. Ant i se rum grading EIA 8. bovis antibody scoret

Antigen dilution 11200 11400 1 1600 11800 111000 111500 112000 114000 118000-1 164000

821 4 strong 19.12.80 positive 9.5 9 7.5 8 6 4.5 5 2.5 0

6721 moderately 6+ standard strong

positive 6 6 3 3 3 1.5 2 1 0

8214 weak

8206 strong

25.1 1.80 positive 1 1 1 1 (1 (1 1 <1 0

negative 1 0 0 0 0 0 0 0 0 Enzyme irnmunoassay

t EIA Score: 0 = negative (less than weak positive standard) (1 = just less than weak positive standard 21 = positive

See text for derivation of EIA score. Reference serums: 1 + mean OD,,, = 0.500 6+ mean OD,,o = 0.950

TABLE 2

Effect of serial dilution of antiserum on EIA score of 4 graded antiserums at antigen dilution 1/400

EIA 8. bovis antibodv score*

Ant i se rum dilution Animal No. Antiserum grading 11100 11200 1 1400 11600 111600 113200 116400 112800

19.12.80 positive 10 7.5 6 2.5 2 1 1 0 8214 strong

6721 moderately 6+ standard strong

8214 weak

8206 strong

positive 6 3 2 1 0 0 0 0

25.1 1.80 positive 1 0 0 0 0 0 0 0

negative 0 0 0 0 0 0 0 0

EIA Score: 0 = negative, EIA absorbance less than weak positive >1 = positive

See text for derivation of EIA score. Reference serums: 1 + mean OD,,, = 0.500 6+ mean OD,,, = 0.950

Australian Veterinary Journal, Vol. 59, November, 1982 137

Results

Effect of Variation of Antigen Concentration To confirm that 1/400 was a suitable dilution of B. bovis

antigen under our conditions, the effect of varying antigen concentration on EIA score was tested with 4 bovine serums diluted 1/100. EIA score was calibrated by comparison with reference serums diluted 1/100 and using 1/400 antigen dilution. The 4 bovine serums were previously shown to have EIA reactions ranging from “strong” negative (from an animal never infected with B. bovis and giving an EIA absorbance just less than the weak positive) to very strong positive.

The EIA scores in Table 1 show that the 2 strong positive serums were positive with antigen dilutions up to 1/4000, while dilutions above 1/800 gave variable results with the weak positive serum. The strong negative serum gave a result which was just equal to a 1 + score at 1/200 but which was negative at higher dilutions of antigen. These results show that 1/400 dilution of antigen gave a good discrimination between weak positive and strong negative and confirmed I / 400 as a suitable dilution of antigen.

Effect o f Variation of Serum Concentration The effect of serial dilution of serum tested at constant

antigen dilution of 1/400 was studied using the same 4 serums. The results in Table 2 show that the strong positive serum had a titre of 1/6400, compared with 1/800 for the moderately strong, 6 + reference serum. The weak positive serum was positive only at 1/100 and the strong negative serum was negative at all dilutions. Therefore, a standard serum dilution of 1/100 was adopted.

TABLE 3

Comparison of EIA and IFAT in determining B. bovis antibody s ta tus

Comparison between tests of total % of Total Fraction

A reement ’!!!3 negative by both tests 1781187 95.2 24 Dositive

1 Susoicious

Disagreement 3 pos i t ive by

EIA bu t neg- ative by IFAT

6 pos i t ive by EIA and sus-

Others 31187 1.6

pected posi- tive by IFAT 61187 3.2

5 25 50 100 200 100 OW 1600 5 25 50 100 200 100 OW 1600

Log. Dilution Reciprocal

FIGURE 1. EIA absorbance of 3 6. bovis reference serums compared with serums from cattle with single or mixed infections of B. bigemina, A. cenfrale and Theileria s p p . H l + Reference serum 1. 148. 6. bovis; -1+ Reference serum 2.6721 (1 1800). 6. bovis; 0-06 + Reference serum. 6721. 6. bovis;(t-O8084 6. bigemina + A. centrale + Thei/eria spp.; D--€ 8233 negative, strongly reacting, con- trol; v - - -V 8251 6. bigemha;+ - - + 8265 6. bigemina + A. centrale + Theileria spp.; A- - -A 8459 A. centrale; ~k; 8460 A. centrale; 0- - - 0 IFA 100 B. bigemina.

14.5

11.8

9.1

EIA

6.4

SCORE

3.7 t . . 1 . 0 1 ‘ 1 ‘ 1 I -4.30 -3.88 -3.45 -3.02 -2.60 -2.17

TITRE (Log,, Dilution)

FIGURE 2. Linear regression of EIA score against EIA titre of 26 serums from cattle infected with K strain 6. bovis.

TABLE 4

EIA titration scores and final titres by EIA and IFAT for B. bovis antibody, of 12 serums from experimentally infected cattle EIA score for 6. bovis antibodv’ Antibodv titre

~~~

Dilution No. 1/90 11270 11810 1 I2430 1 I7290 1121,870 EIA IFAT

1.5 0 0 0 0 0 90 30 2 0 0 0 0 0 90 10 2 0 0 0 0 0 90 30

2.5 0 0 0 0 0 90 30 3 0 0 0 0 0 90 10

4.5 3.5 1.5 0 0 0 810 810 6 3.5 3.5 1.5 1 0 7290 2430

7.5 4.5 3 2.5 1 0 7290 81 0 10 8 7.5 5 3 1 21,870 7290 12 9 8.5 6.5 2.5 0 7290 2430

12.5 9.5 7.5 6 3.5 0 7290 2430 12.5 10 9 7.5 4 1 21,870 2430

EIA score 0 = negative 21 = positive

See text for derivation of EIA score. Reference serums: 1 + mean ODdoo = 0.350 6 + mean ODdOo = 0.850

138 Australian Veterinary Journal, Vol . 59, November, 19R2

TABLE 5

Course of antibody response, as measured by EIA, In cattle Infected with B. bovis and challenged heterologously 71 days later

~~~~~

EIA score for 6. bovis antibodv' (mean 2 S.E.)t Day and strain Number of of initial Day and strain Days post infection

cattle infection of challenge 0 14 20 27 41 55 69 8a

5 0 71 0 5.8ka 8 .0 ib 7 . 5 i b 9 .4 icd 8.6ibc 3.6*bc k strain R strain 0.60 0.57 0.67 0.76 0.81 0.46 0.45

5 71 - N D N D N D N D N D N D 0 5 . 6 i R strain 0.96

N D = not determined EIA score: 0 = negative 21 = positive See text for derivation of EIA score. Reference serums: 1 + mean ODdoo = 0.590 6 + mean OD., =, 1.090 Means with diyfering superscripts were significantly different (~(0.05) . t

Specificity for B. bovis Antiserums The cross reactions between B. bovis antigen and anti-

serums from cattle with single or mixed infections of B. bigemina, A . centrale, and an Australian species of Theileria (formerly thought to be T . mutans, Uilenberg et al, 1977), were tested over a range of serum dilutions. As shown in Figure 1, although there was a measurable cross reaction with all serums, these reactions at each dilution tested were less than the 1 + reference serums, either 148 or 6721 (1/800) and therefore were never high enough to give false positive readings. There were no additive effects of serums from mixed infections since animals with all 3 parasites were negative.

Correlation between EIA Score and EIA Titre Linear regression of the EIA score against EIA titre of 26

serums from cattle infected with K strain B. bovis was calculated. The resultant regression line, shown in Figure 2, had a correlation coefficient (r) of 0.941 which was statisti- cally significant (p<O.OI).

Comparison o f EIA and IFAT in Determining Antibody Status to B. bovis

One hundred and eighty seven serums from 109 cattle (85 from tick-free pastures and 24 Bos taurus x Bos indicus cross cattle from lightly tick-infested pastures), were screened by both EIA and IFAT to determine the correlation between the 2 methods. The results in Table 3 show that there was agreement between the tests for 95.2% of serum samples, disagreement for 1.6% and 3.2% were positive by EIA and suspected positive (neither definitely positive nor definitely negative) by IFAT.

Comparison o f Sensitivity of EIA and IFAT Sensitivities of EIA and IFAT were compared by titrating

12 serums which were positive for B. bovis. Tripling dilutions

50 100 150 200 250 300 Time after Infection (days) .

FIGURE 3. Duration of antibody response in one steer meas- ured by both EIA and IFAT for 268 days pi;- EIA score; u IFAT score.

Australian Veterinary Journal, Vol. 59, November, 1982

were used, starting at 1/10 for IFAT and 1/90 for EIA. The results on Table 4 show that for 11 of the serums, the EIA was more sensitive than the IFAT by at least one dilution factor.

Duration o f Antibody Response Measured by EIA and IFA T

A calf about one month of age was inoculated with U strain B. bovis and serum collection begun 4 days later when parasites were detected. Serum samples were collected for 268 days post infection (pi) and B. bovis antibody levels estimated by both EIA and IFAT. As shown in Figure 3, serum was positive by both methods on day 18 pi and remained positive for 268 days pi by both methods.

Course o f Antibody Response Measured by EIA in Animals Experimentally Infected with B. Bovis and Heterologously Challenged

Five cattle were inoculated subcutaneously with K strain B. bovis on day 0 . On day 71 pi these cattle and another' group of previously uninfected cattle were inoculated with R strain B. bovis. Mean antibody scores of the cattle are shown in Table 5 . Positive EIA scores were found by day 14 pi in cattle infected on day 0. Seventeen days after heterol- ogous challenge there was a significant (~(0.05) increase in the mean EIA score of the 5 immune cattle while the control group then had a mean EIA score similar to group one on day 14 pi.

Discussion A microplate EIA method is described for detecting and

measuring IgG antibody to B. bovis in cattle serum. B. bovis antigen was prepared from infected bovine blood and optimum antigen dilution was 1/400 at serum dilution 1/100. Reference serums were used to define the absorbance thresh- old between B. bovis positive and negative serums and to calibrate the EIA absorbance on a scale from one to about 14 (EIA score), thereby minimising day to day fluctuations in results due to variations in materials or method. The EIA score, measured at 1/100 serum dilution, was shown to correlate well with EIA titre.

Specificity of the EIA for B. bovis was shown by the lack of significant cross reaction between B. bovis antigen and serums from cattle with single and mixed infections of B. bigemina, A . centrale and Theileria spp.

When EIA and IFAT were compared, there was agreement between methods for 95.2% of serums tested and disagree- ment for only 1.6%. The remaining 3.2% were positive by EIA and suspected positive by IFAT. By comparing the B. bovis antibody titre of positive serums by both EIA and IFAT, the former test was shown to be the more sensitive.

Positive EIA scores were found in experimentally infected cattle by day 14 pi and for up to 268 days pi by both methods. Following heterologous strain challenge there was

139

a significant (~(0.05) increase in the mean EIA score of immune cattle.

EIA methods have been described for other Babesia species, B. canis, B. gibsoni (Weiland and Kratzer 1979) and B. divergens (Purnell el a1 1976; Bidwell et a1 1979) and B. major (Bidwell et a1 1979). The latter workers found positive reactions for B. divergens by EIA and IFAT in experimentally infected, splenectomised, calves from day 13 pi to day 216 pi, findings which are similar to those reported here for B. bovis.

A novel refinement of the EIA technique described here was the use of reference serums to define objectively the difference between positive and negative serums, and to calibrate EIA absorbance as a score over a wide range of antibody levels. Absorbance values for reference serums were found to vary between estimations and we considered that inclusion of these serums with each microplate was essential. Expression of antibody levels in test serums as EIA scores rather than as absorbance values simplified comparisons between results obtained at different times and with different microplates.

Although the EIA score may not give as accurate a measurement of relative antibody levels as an EIA titre, there was a highly significant correlation between the EIA score and EIA titre. This and other data, such as the significant increase in mean EIA score of immune cattle following heterologous challenge, showed that the EIA score is a useful index of B. bovis antibody levels. An advantage, therefore, of the EIA score is that it gives a quantitative estimate of B. bovis antibody levels from a single dilution of test serum.

Despite the use of the same reference serums and a serum dilution of 1/100 compared to 1/30 for the IFAT, the EIA found a few serums positive which were negative by IFAT. This may have been due to the greater sensitivity of the EIA as shown by titration of positive serums. However, the possibility that the EIA gives a small proportion (less than 2%) of false positive readings cannot be ruled out without further investigation.

The EIA test was found to have the advantages over the IFAT of being a n objective test, of giving a quantitative

result and of being more sensitive. These findings were similar to those of Bidwell et a1 (1979), who found the EIA preferable to the IFAT for B. divergens because there was less operator error and stress. A possible disadvantage of the method as described here, is that i t takes longer to complete than the IFAT. For example, starting the EIA in the afternoon, with an intervening overnight incubation, a result may be obtained the next morning, whereas an IFAT result may be obtained the same afternoon.

Acknowledgments The authors thank R. J . Dalgleish for helpful discussion and F.

Duncalfe for statistical analyses. This work was supported by the Queensland Department of Primary Industries and the Australian Meat Research Committee.

References Bidwell, D. E., Joyner, L. P. and Purnell, K. E. (1979) - Trans.

Roy. SOC. trop. med. hyg. 73: 105. Callow, L. L., McGregor, W. Parker, R . J . and Dalgleish. R . J .

(1074) - Ausr. vet. J . 50: 6. Callow, L. L. (1977) - Vaccination against bovine babesiosis. In

Immunity to Blood Parasites of Animals and Man. (Eds Miller, L. H . , Pino, S . A. and McKelvey, Jr., J. J.) p.121. Plenum Publishing Corp., New York.

Dodge, J. T., Mitchell, C . and Hanahan, D . J . (1963) - Arch. Bichem. Biophys. 100: 119.

Engvall, E. and Perlmann, P. (1972) - J . Immunol. 109: 129. Mahonev. D . F. (1967) - EXO. Parasit. 20: 232. Purnell,<R. E. , Hendry, D . F.', Bidwell, D . W. and Turp, P . (1976)

Snedecor, P . E. and Cochran, W. G . (1967) - Statistical Methods, - Vet. Rec. 99: 102.

6th edn, Iowa State University Press, Ames, Iowa.

M. P. (1980) - J . clin. Microbiol. 11: 499.

(1977) - Aust. vet. J . 53: 271.

0. 53: 55.

Wochenschr. 92: 398.

Thoen, C . O. , Blackburn, B. , Mills, K., Lomme, J . and Hopkins,

Uilenberg, G. , Mpangala, C. , McGregor, W. and Callow, L . L.

Voller, A. , Bidwell, D. F. and Bartlett, A. (1976) - Bull. W. H .

Weiland, Von, G . and Kratzer, I . (1979) - Berl. Munch. Tierarz.

(Accepted for publication I5 July 1982)

Experimental production of dermatitis in sheep with Pseudomonas aeruginosa

D. H. BURRELL*, G. C. MERRITTI, J. E. WATTSt and K. H . WALKERt

SUMMARY: The attachment, to sheep skin, for 4 days, of control wool pads saturated with sterile culture medium which contained a bacteriostat, induced only a mild dermatitis, whereas wool pads saturated with Pseudomonas aeruginosa culture induced a subacute dermatitis characterised by scaling, microabscess formation and seropurulent exudate. Changes similar to the latter were observed in skin affected by natural fleece-rot which developed spontaneously after 7 days of artificial wetting and in which P. aeruginosa was the predominant species of bacteria. An exacerbatory, if not causal, role for this organism is suggested in the development of the dermatitis associated with fleece-rot and in the exudation of seropurulent material, a step essential in the development of body strike. Aust. J . vet. 59:

Introduction

obtained from fleece-rot lesions, and clinical observation of the development, in fleece-rot, of a superficial inflammatory condition with seropurulent exudation and production of a pronounced d o u r were described by Seddon (1937). Bel- schner (1937), in addition to recording that bacteria actively

growing in the fleece produce putrefactive odours attractive The abundance of Pseudomonas aeruginosa in wool to the blowfly, postulated that multiplication of bacteria on

the skin, leading to a superficial dermatitis with consequent serious exudation, was the early sequence of events in the development of fleece-rot. Hayman (1953) showed that fleece- rot was invariably associated with a mild dermatitis and Mackerras (1936) concluded that dermatitis must be present for blowfly strike of fleece-rot lesions to occur.

The predominance of P. aeruginosa in fleece-rot lesions has been quantitatively confirmed (Merr i t t and Watts 1978a) and the attractiveness of its volatile products for oviposition by the blowfly, Lucilia cuprina, demonstrated (Merritt and

CSlRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, P.O., Glebe, New South Wales 2037

t Department of Agriculture, Veterinary Research Station, Glenfield, New South Wales 2167

140 Australian Veterinary Journal, Vol. 59, November, 1982