Veterinary Parasitology, 24 (1987) 195-202 195 Elsevier Science Publishers B.V., AmsterdAm - - Printed in The Netherlands

Serodiagnosis of Porcine Cysticercosis by Enzyme- Linked Immunosorbent Assay (ELISA) using Fractionated Antigens

D. KUMAR and S.N.S. GAUR

Department of Parasitology, College of Veterinary Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar-263145 (India)

(Accepted for publication 16 July 1986)

ABSTRACT

Kumar, D. and Gaur, S.N.S., 1987. Serodiagnosis of porcine cysticercosis by enzyme-linkedimmu- nosorbent assay (ELISA) using fractionated antigens. Vet. Parasitol., 24: 195-202.

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. soliura larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, r~spectively.

INTRODUCTION

The enzyme-linked immunosorbent assay (ELISA) of Engvall and Perl- mann (1971, 1972) has been used in the diagnosis and serology of a number of human diseases (Voller et al., 1976) and animal diseases (Ruitenburg et al., 1976; Craig and Rickard, 1981; Geerts et al., 1981; Gamble et al., 1983). Aram- bulo et al. (1978) described the micro-ELISA test for the serodiagnosis of human cysticercosis. Espinoza et al. (1982) used the ELISA and immunoelec- trophoresis to test for human cysticercosis and observed false-positive results with both tests. The use of ELISA in the serodiagnosis of porcine cysticercosis has not so far been investigated since serological tests provide a specific ante- mortem diagnosis. The present study was undertaken to investigate the sen- sitivity and specificity of this test in naturally and experimentally infected cases of porcine cysticercosis.

0304-4017/87/$03.50 © 1987 Elsevier Science Publishers B.V.

196

MATERIALS AND METHODS

Antigen preparations

Fresh T. solium cysticerci collected from pigs at slaughter were washed in normal saline and host tissues were gently removed. The scoleces were sepa- rated, washed in 0.15 M NaC1 and frozen in 0.15 NaC1 at -20°C. They were removed after 24 h, well triturated in 0.15 M NaC1 (pH 7.0), homogenized in a glass homogenizer and centrifuged at 10 000 rpm for 30 min in a refrigerated centrifuge. The supernatant was sonicated for 5 min in an MSE 150 W ultra- sonic disintegrator peak to peak 15/~m, taking care that the temperature of the suspension did not rise above 2 ° C.

Sonicated antigen was then dialysed in polyethylene glycol (6000) for 6 h at 4 ° C and a few drops of merthiolate (1:10 000) were added before storage at -20°C.

Fractionation of T. solium larval scolex ( Exs ) antigen by Sephadex column chromatography

Ascending flow gel filtration chromatography was performed on a 100 >< 2.5 cm column of Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Swe- den) equilibrated with PBS (pH 7.2). Five ml of T. solium larval scolex anti- gen was applied to the column at a flow rate of 18 ml h - 1. Aliquots of 5 ml were collected and the absorbance of each fraction was measured at 280 nm. Three peaks were pooled, concentrated in polyethylene glycol and stored at - 2 0 ° C until used. The protein content of these antigens was estimated according to the method of Lowry et al. (1951).

Preparation of rabbit hyperimmune sera

Rabbits were immunized against antigens of larval T. solium and T. hyda- tigena scoleces using five injections, with doses gradually increasing from 1.0 ml to 3 ml, given at intervals of 8 days. The rabbits were bled intracardially on Day 7 after the last injection; the serum was separated under strict sterile con- ditions and stored at - 20 ° C after adding a few drops of merthiolate.

Other sera samples

Naturally infected pigs Blood samples were randomly collected from conventionally raised pigs which

were subsequently slaughtered and confirmed to be positive or negative for T. solium cysticercosis at necropsy. Negativity of these animals was further con- firmed by examining sections of the tongue, liver, heart and skeletal muscles

197

using a magnifying glass. A total of 20 and 11 serum samples were collected from T. solium-positive and -negative pigs, respectively. Serum samples from positive animals also infected with Ascaris suum, Stephanurus dentatus, Fas- ciolopsis buski, larval forms of Taenia hydatigena and Echinococcus granulosus were discarded, along with other doubtful samples.

Experimentally infected pigs Blood samples from four piglets experimentally infected with 15 000 eggs of

T. solium were obtained. Sera were stored at - 2 0 ° C after adding a few drops of merthiolate solution (1:10 000).

Other helminth infections Serum samples were obtained from pigs naturally infected with A. suum and

larval forms of T. hydatigena and E. granulosus infections.

Enzyme-linked immunosorbent assay ( Micro-ELISA )

Technique The procedure adopted for the ELISA test was that described by Voller et

al. (1976) with slight modifications. The test was standardized with hyper- immune rabbit sera and further evaluated with pig sera. Dilutions of test sera from 1:5-1:100 were prepared; various dilutions of antigen were also tested to obtain opt imum results. Antigen at a 1:10 dilution in 0.1 M sodium carbonate buffer (pH 9.6), equivalent to 2.5 mg protein ml-1, gave opt imum and consis- tent results. The optimum incubation time of plates was determined to be 1.5 h.

Titration of antigens with hyperimmune rabbit sera Ten-fold serial dilutions of each test antigen, from 10 -1 to 10 -7 , were pre-

pared in carbonate bicarbonate buffer of pH 9.6 and 0.1 ml of each dilution was transferred to wells of polystyrene microtitre substrate plates. The plates were sensitized overnight at 4 ° C, emptied and washed three times with phos- phate buffer saline-Tween-20 (PBS-tween), pH 7.2. Hyperimmune rabbit serum samples were diluted 1:50 in phosphate buffer saline, pH 7.4, containing 0.05% Tween-20 and 1% bovine serum albumin (BSA); 0.1 ml of this diluted sample was put into the wells. Conjugate and substrate control wells were left unfilled and plates were incubated at 37 ° C for 1.5 h, emptied and washed three times as above. Alkaline phosphatase-conjugated anti-rabbit IgG (M/s Sigma Chemicals Company, St. Louis, MO, U.S.A. ) was diluted 1:1000 in Tris buffer (pH 8.0) and 0.1 ml of this diluted conjugate was put into all the wells. After incubation for 1.5 h at 37 ° C the untreated conjugate was removed by washing three times with PBS-Tween-20 (pH 7.2). 0.1 ml of freshly prepared 0.1% solution ofp-ni trophenyl phosphate disodium salt in glycine buffer, pH 10.4,

198

was added to each well in subdued light. The plates were incubated for 30 min at 37 °C in a dark chamber and readings were recorded after the reaction had been stopped with 0.05 ml of I N NaOH.

Substrate, conjugate control, antigen control and serum controls were also run.

Titration of rabbit hyperimmune sera 0.1 ml of antigen at 1:10 dilution was used for this purpose and 10-fold serial

dilutions of hyperimmune rabbit sera, from 10-1 and 10-7, were prepared in PBS-Tween-20. The same test procedure was adopted as for the titration of antigen.

Titration of pig sera Experimentally and naturally infected pig sera was titrated against scolex,

1st and 2nd peak antigens using 0.1 ml of each antigen at a 1:10 dilution and 10-fold serial dilutions of infected pig sera, from 10-1 to 10-7, in PBS-Tween. The same test procedure was adopted as for the titration of rabbit hyperim- mune sera, except that anti-swine IgG conjugated to horse radish peroxidase ( Kirkegaard and Perry Laboratories, Gaithersburg, MD, U.S.A. ) was diluted to 1:300 in PBS-Tween and 1% BSA and allowed to react for 1.5 h at 37°C. The untreated conjugate was removed by washing three times with PBS-Tween (pH 7.2). Finally 0.1 ml of a freshly prepared solution of 0.04% orthopheny- lenediamine in phosphate citrate buffer, pH 5.0, was added. The plate was incubated at 37 ° C for 15 min, the reaction was stopped with 0.05 ml 2 M H2SO4 and the absorbance values were obtained at 450 nm after preparation of a 1:100 dilution from each well.

The sensitivity of the test for T. solium was evaluated with serum samples collected from four experimentally and 20 naturally infected pigs. The speci- ficity of the test was evaluated with the sera of 11 uninfected pigs, five pigs infected with A. suum, four with larval forms of T. hydatigena and six infected with E. granulosus. For this purpose a 1:10 dilution of antigen and a 1:50 dilu- tion of test sera were used. The reagents and procedure were as in the titration of infected pig sera. Serum samples with absorbance values more than double those of normal serum were considered positive.

RESULTS AND DISCUSSION

Titration of antigens

A titre of 106 was obtained using T. solium larval scolex and 1st peak antigens with hyperimmune rabbit sera raised against T. solium larval scolex antigen. Titration with 2nd and 3rd peak antigens gave titres of 105 and 103, respectively.

199

3 9_ o 0.5

0.45

:~ 0,4

0.35

3 0.3

~, o.25

0.2

E 0.15 ¢:

0.1 -4" 0,05

0 d o

Scoiex

O 0 0

• o o

o :

• e ~

~ o o • • e o l ~ e

o

I st peak

o o •

:o e o

o o o

o oe@ • • • e e ®

e

2 nd peak

o o •

: o

::o t

e o e °°® * : ; : : o ,

• Infected p,g sero • Unlnfect¢ci p,g sera

Fig. 1. Comparison of micro-ELISA absorbance values at 450 nm obtained with saline extract of T. solium larval scolex antigen and its fractionated 1st and 2nd peak antigens against T. solium larval infected pig sera.

Titration o[ hyperimmune rabbit sera

A titre of 104 was recorded with hyperimmune rabbit sera raised against T. solium larval scolex when titrated with scolex and 1st peak antigens. With 2nd and 3rd peak antigens, the titres were 103 and 10 I, respectively.

Titration of pig antisera

Titres of 103-105 were observed in infected pig sera with 1st peak antigen, titres of 102-104 and 101-103 were obtained with scolex and 2nd peak antigens, respectively.

Sensitivity

The sensitivity of this test was evaluated in experimentally and naturally infected pig sera, and the comparison of micro-ELISA absorbance values at 450 nm with these antigens and pig sera is illustrated in Fig. 1. All four exper- imentally infected animals showed positive reactions with scolex and 1st peak antigens. However, one animal (25%) gave false-negative results with 2nd peak antigen. In 20 naturally infected pig sera 5 and 10% false-negative reac- tions were found with 1st peak and scolex antigens, respectively. However, 30% false-negative reactions occurred with 2nd peak antigen.

An overall sensitivity of 95.8% was found with the 1st peak antigen, whereas scolex antigen gave 91.6% sensitivity. The lowest sensitivity (70.8%) was given

TA

BL

E I

Sen

siti

vity

of

EL

ISA

tes

t in

por

cin

e cy

stic

erco

sis

usi

ng

Tae

nia

soli

um l

arva

l sco

lex

and

its

frac

tion

ated

1st

an

d 2n

d pe

ak a

nti

gen

s

An

tige

n

use

d E

xper

imen

tall

y in

fect

ed p

igs

No.

of

Pos

itiv

e F

alse

se

ra

reac

tion

n

ega-

te

sted

ti

ve

Sen

siti

vity

(o

/o)

Nat

ura

lly

infe

cted

pig

s O

vera

ll

sen

siti

vity

N

o. o

f P

osit

ive

Fal

se

Sen

siti

vity

(S

o)

sera

re

acti

on

neg

a-

(So)

te

sted

ti

ve

Sco

lex

4 4

0 10

0 20

18

2

90

91.6

1st p

eak

4

4 0

100

20

19

1 95

95

.8

2ndp

eak

4

3 1

75

20

14

6 70

70

.8

TA

BL

E I

I

Spe

cifi

city

of

EL

ISA

tes

t in

por

cine

cy

stic

erco

sis

with

T

aeni

a so

lium

lar

val

scol

ex

and

its f

ract

iona

ted

1st

and

peak

an

tigen

s

Ant

igen

us

ed

Fals

e po

sitiv

e C

ross

re

actio

n Sp

ecif

icity

(76)

H

ealth

y T

. hy

dati

gena

lar

vae

E.

gran

ulos

us

A.

suum

un

infe

cted

(1

1) a

(4

) la

rvae

(5

) (6

)

Sco

lex

1 1

0 0

92.3

1s

t pea

k 1

0 0

0 96

.2

2ndp

eak

0 1

0 1

92.3

“Fig

ures

in

par

enth

eses

sh

ow

the

tota

l nu

mbe

r of

pig

ser

a te

sted

.

201

with 2nd peak antigen (Table I) . High sensitivity (59.0 and 82.5% ) was also recorded with this test by Feigner (1978) and Dottorini et al. (1981) in human echinococcosis cases. In contrast, Geerts et al. (1981) showed poor sensitivity (37.5%) of this test in bovine cysticercosis, using T. crassiceps metacestode antigen. As pointed out by Dottorini et al. (1981) this could be due to the heterologous antigen used. Higher sensitivity against human hydatid disease was obtained with human E. granulosus cyst fluid as the source of antigen in place of E. granulosus cyst fluid of sheep origin ( Felgner, 1978). Pauluzzi et al. (1981) also recorded that human E. granulosus cyst fluid had a higher specific antigenic activity than that of sheep.

Specificity

In the present investigations one of 11 uninfected pig sera (9.09%) gave a false-positive result with scolex and 1st peak antigens. No false-positive reac- tion was observed with 2nd peak antigen. One cross-reaction was recorded out of the four animals infected with T. hydatigena cysticercosis when tested with scolex and 2nd peak antigens. One A. suum-infected pig serum cross-reacted with 2nd peak antigen. However, no cross-reaction was recorded with 1st peak antigen and this also had the highest specificity (96.2%; Table II) . The cross- reaction in serum from sheep infected with T. hydatigena cysticercosis and cattle harbouring natural infection by Fasciola hepatica was also recorded by Geerts et al. (1981). They observed 4.3% false-positive results in T. saginata cysticercosis.

ACKNOWLEDGEMENTS

The authors are grateful to the Dean, College of Veterinary Sciences, Head, Parasitology and Director of Research for providing the necessary facilities. Financial assistance from the Indian Council of Agricultural Research is highly acknowledged.

REFERENCES

Arambulo, P.V., III, Walls, K.W., Bullock, S. and Kagan, I.G., 1978. Serodiagnosis of human cysticercosis by microplate enzyme-linked immunospecific assay (ELISA). Acta Tropica, 35: 63-67.

Craig, P.S. and Rickard, M.D., 1981. Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme- linked immunosorbent assay (ELISA). Int. J. Parasitol., 11: 441-449.

Dottorini, S., Tassi, C. and Baldelli, F., 1981. ELISA (enzyme-linked immunosorbent assay} for diagnosis of human hydatid disease. Boll. Ist. Sieroter. Milanese, 60: 137-143.

EngvaU, E. and Perlmann, P., 1971. Enzyme-linked immunosorbent assay ( ELISA ). Quantitative assay of immunoglobulin G. Immunochemistry, 8: 871-874.

202

Engvall, E. and Perlmann, P., 1972. Ezyme-linked immunosorbent assay (ELISA). II. Quanti- tation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J. Immunol., 109: 129-135.

Espinoza, B., Flisser, A., Plancarte, A. and Larralde, C., 1982. Immunodiagnosis of human cysti- cercosis: ELISA and immunoelectrophoresis. In: A. Flisser, K. Willms, J.P. Laclette, C. Lar- ralde, C. Ridaura and F. Beltran (Editors). Cysticercosis: Present State of Knowledge and Perspectives. Academic Press, New York, London, pp. 163-170.

Felgner, P., 1978. Antibody activity in stick ELISA as compared to other quantitative immuno- logical tests in sera of echinococcosis cases. Tropenmed. Parasitol., 29: 417-422.

Gamble, H.R., Anderson, W.R., Graham, C.E. and Murrell, K.D., 1983. Diagnosis of swine trichi- nosis by enzyme-linked immunosorbent assay (ELISA) using an excretory-secretory antigen. Vet. Parasitol., 13: 349-361.

Geerts, S., Kumar, V., Ceulemans, F. and Mortelmans, J., 1981. Serodiagnosis of Taenia saginata cysticercosis in experimentally and naturally infected cattle by enzyme-linked immunosorbent assay. Res. Vet. Sci., 30: 288-293.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.

Pauluzzi, S., Baldelli, F., Dottorini, S. and Tassi, C., 1981. Standardizzazione quantitativa del metodo ELISA nell'idatidosi umana. Boll. Ist Sieroter. Milanese, 60: 144-149.

Ruitenberg, E.J., Steerenberg, P.A., Brosi, B.J.M. and Buys, J., 1976. Reliability of the enzyme- linked immunosorbent assay (ELISA) for the diagnosis of Trichinella spiralis in convention- ally raised pigs. J. Immunol. Methods, 10: 67-83.

Voller, A., Bidwell, D.E. and Bartlett, A., 1976. Enzyme immunoassay in diagnostic medicine. Bull. WHO, 53: 55-56.