A purified Fasciola gigantica worm antigen for the serodiagnosis of human fascioliasis

TRANSACTIONSor THE ROYAL SOCIETY OF TROPICALMEDICINEAND HYGIENE (1991) 85, 535-537

A purified Fasciola fascioliasis F. G. Youssef

gigantica

and N. S. Mansour

worm

antigen

535

for the serodiagnosis

of human

BasicScienceDivision, US Naval Medical ResearchUnit No. 3, Cairo,

Emt

Abstract

Partially purified Fasciolagiganticaworm antigens were evaluatedin an enzyme-linked immunosorbent assay(ELISA) for the specificserologicaldiagnosisof fascioliasis.The diagnostic potential of 10 antigen fractions derived from gel-filtration peaks(II & III) were evaluatedindividually or in combinationagainst a panel of admissionseraobtained from 50 patients subsequentlydiagnosedas having a variety of systemic narasiticdiseases, andfrom 10healthv volunteers with- no recent history of parasitic infections. Fractions 2 and 10 were highly specific for Fasciolu infection and failed to reactwith serafrom any natient with other parasitic diseasesor from con&&. All other fractions exhibited reactivity with the majority of sera from the fascioliasispatients, but also with someserafrom patientsinfected with other parasites. Basedon theseresults, the ELISA, asoutlined here, shouldbe consideredasa rapid and-specifictechnique for the diaanosis of fascioliasisand also as an epidemiologiialsurveillancetechniquein areasendemic for F. giganticainfection. Introduction

Partially purified antigen from Fasciola gigantica worms has been used in a counter-immunoelectrophoresis(CIEP) assayfor the specificdiagnosisof human fascioliasisin Egypt (MANSOUR et al., 1983). This partially purified antigen was derived from a portion of peak II and all of peak III resulting from gel filtration (SephadexG-200@)of a crude extract of adult F. giganticaworms. When this partially purified Fasciolaworm preparation was used in an enzymelinked immunosorbentassay(ELISA), it reactedwith serafrom fascioliasispatientsbut alsogave signiiicant cross reactions with some sera from patients with other parasitic diseases,such as schistosomiasis, amoebiasis andechinococcosis. The nresentstudv was undertaken to prepare a more specific F. gig&tica worm antiaen which would be hiahlv reactive in the ELISA wirh serafrom humanfasc?ohasis patientsbut non-reactive with sera from patients with other systemicparasitic infections or with control sera. Materials

and Methods

Antigens Partially purified F. giganticaworm antigens(peaks II and III as separatedby gel filtration on Sephadex G-200@)were preparedas describedby MANSOUR et al. (1983). Column eluates were collected as 10 aliquots of 5 ml each. They were savedfor further Requests for offprints should he addressed to Research Publications Branch, US Naval Medical Research Unit No. 3 (NAMRU-3), FPO New York 09527-1600, USA.

evaluation as sensitiveand specificantigensfor the serologicaldiagnosisof F. gigunricainfection using the ELISA procedure. Sera Serawereobtained, upon admissionto hosnitaland before treatment, from the following patient groups. (i) Twentv hosnitalnatientswith the tvnical clinical symptoms of fa&ioliasis, passingFas~~olaeggs in their stools,positive serologyby CIEP, and positive clinical responseto subsequentemetinehydrochloride therapy. 100 ld of eachof theseserawerealsopooled and used as a positive control. (ii) Eighteen schistosomiasis patients who were negativefor Fascioluinfection by parasitological(stool examination) and serological(CIEP) methods. Of thesepatients, 6 were excreting Schistosoma haematobiumeggsin the urine, 6 wereexcretingS. mansoni in. stool, and the remaining6 wereexcreting eggsof both species. (iii) Eiaht patients with amoebic liver abscess. diagnosed-bydlinical criteria and confirmed serologi: callv usina CIEP and indirect haemanelutination -(IHA). *(iv) Four patients with hydatid diseasediagnosed &;cally and confirmed serologicallyby CIEP and Negative control sera were obtained from 10 healthy volunteerswho were negativeat 3 successive stool examinationsfor these various parasites. Sodium dodecyl sulphute-polyacrylamidegel electro&oresis(SDS-PAGE) SDS-PAGE gel analysis of F. giguntica antigen fractions was carried out in a discontinuousbuffer system as describedby LAEMMLI (1970). Antigens were dialysed against phosphate-buffered saline (pH 7-O)and concentratedto approximately4 mg of protein per ml. Parasitologicalexaminationof stool Stools

were

examined

after

mertbiolate-iodine-

formaldehyde-ether concentration (BLAGG et al., 1955). Diagnosisof Fasciolainfection was basedon egg morphology and size (WATANABE, 1965). Counter-immunoelectrophoresis This test was carried out as described by MANSOUR

et al. (1983).

Enzyme-linkedimmunosorbent assay The ELISA was carried out as described bv G~TTSTEIN et al. (1983). Antigen fractions were diluted to a concentrationof 50 ug of protein per ml in the coatingbuffer. All patient andcontrol serawere

536 diluted 1:50 in washing buffer and 100 ~1 were added to each well after blockinn. The limit for discriminating negative from positive results was determined by the mean value of the negative controls +2 standard deviations.

(

Results

Size fractionation of a homogenateof adult F. (30 mg protein) on a Sephadex G-200@ column yielded 4 distinct protein peaks (Fig. 1). et al., 1983),peaks From a previousstudy (MANSOUR II and III were known to contain important F. gigunticu antigens. Consequently, the 10 fractions included in these 2 peakswere selectedfor further biochemical characterization and evaluation as diagnosticantigens for fascioliasis. The protein profiles of these 10 fractions after electrophoreticseparationby SDS-PAGE are shown in Fig. 2. Proteins with molecularweights ranging between 10 and 66 kDa were present in the first 8 fractions, while proteinsin the M kDa rangewere presentedin fractions 9 and 10. All fractions shared prominent bandsat 12, 14,29,31 and 40 kDa, while the first 8 fractionssharedadditionalprominentbands at 42, 45, 48, 54 and 58 kDa which were not detectablein fractions 9 or 10. Fractions 2 and 10 lacked bandsat 37, 54, 60 and 66 kDa which were presentin most of the dther fractions and in peaksII and III (Fig. 2). When the 10fractions wereevaluatedin an ELISA for serologicaldiagnosisof fascioliasis(Table l), it wasnoted that the first 6 fractions diagnoseda larger percentageof the fascioliasispatients (75-90%) than the remaining 4 fractions (40-60%). The fractions within each of these 2 groups were very similar in their diagnosisof fascioliasiscases.With the exception of fractions 2 and 10, the other fractions cross-reactedto varying degreeswith someserafrom patientswith other systemicparasiticinfections.Fraction 2 diagnosed18 of the 20 Fusciolu caseswhile fraction 10 diagnosed12. When fractions 2 and 10 were pooled,they diagnosedall 20 fascioliasis patients without any false positive results with sera from healthy controlsor from patientswith other parasitic infections.The results of comparing the relative diagnostic values of crude (unfractionated worm homogenate),partially purified F. gigunticu antigens (peaks II and III, Fig. 1) and the pooled antigen (fractions2 and 10) in ELISA and CIEP are shownin Table 2. The pooledantigen preparation gave 100% sensitivity and specificity for fascioliasisin both serologicaltests,while the other antigen preparations demonstratedsomenon-specificcrossreactionswith sera from patients infected with other parasites. gigunzicu

Discussion

When partially purified F. gigunticu antigens(peaks II and III) were usedin CIEP, a highly specifictest resultedexhibiting essentiallyno crossreactivity with seraof patients with protozoa1or other helminthic et al., 1983).When thesesame infections(MANSOUR antigens were used in an ELISA, cross reactions occurred with sera from patients with confirmed amoebiasis,schistosomiasis and echinococcosis infections. Electrophoretic analysisof the 10 fractions of peaks II and III indicated that fractions 2 and 10

50

60

70

60

90

100

110

VOLUME

120

130

140

I50

160

170

I60

,mL,

Fig. 1, Elution profile of F. gigantica adult worm homogenate Sephadex G-200@. The 10 numbered fractions constituting trailing portion of peak II and all of peak III were saved subsequently evaluated as diagnostic antigens in an ELISA for serological diagnosis of fascioliasis. Mr12

66.200-

3

45

8

on the and the

7891Om

2:‘: ‘"‘

Fig. 2. SDS-PAGE analysis of F. gigantica proteins present in fractions l-10 resulting from filtration of adult worm homogenate on a Sephadex G-200@ column (Fig. 1). Protein fractions were separated by electrophoresis on lO-20% gradient gels and protein bands were visualized by Coomassie blue staining. Relative molecular mass (M,) standards (lane 1) and peaks II and III (combined) from the G-200 column eluate (lane 12) were included for reference and comparative purposes.

lacked protein bandsin the 37, 54, 60 and 66 kDa ranges,which may have contributed significantly to the cross reactivity problems the other 8 fractions demonstrated.Usingfractions 2 and 10resultedin no non-specificreaction in either the ELISA or CIEP. SANTIAGO & HILLYER (1986) reported that F. heputicu worm antigensof < 14 kDa molecularweight were recognizedas early as 11 d after infection in experimentallyinfected mice, and that proteinsin the 2&23 and 31-33 kDa range reacted strongly with serafrom infected patients but not with serafrom patientswith other parasitic diseases. These authors alsoreportedthat cross-reactions were associated with proteins in the 10-12 and 30 kDa range. Although antigensin the 10-12 kDa range were present in fractions2 and 10 of F. gigantica wormsin this study, they appeared to have no adverse effect on the specificityof the ELISA. HILLYER & GALANES (1988) identified F. heputicu excretory-secretoryworm antigensin the 17 and 63 kDa range; serodiagnosis of fascioliasiswas specific with the 17 kDa antigen, while the 63 kDa antigen cross-reactedwith sera from humansinfected with S. munsoni and Trichidlu spiralis. The greatsimilarity betweenF. gigantica and F. heputicu worm protein bandingin the variouskDa rangessuggests diagnosticreagentsfrom thoseworms

537 Table 1. Specilicity and sensitivity of the enzyme-linked partially puritied F. gigantica worm antigens

Clinical/ serological diagnosis’ Fascioliasis S. mansoni

Total no. of patients 20

immuaosorbent

t 6 :

S. haematobium S. mansoni & S. haematobium

2 18

1 17 i

:

i

0 :

assay

for the diagnosis

of fascioliasis

Number of patients positiveb Fractions 3 4 5 6 7 8 17 15 17 16 i : :

:

i

i

i

i

:

using

9 12

10 12 i

0

0

8

1

1

1

8 : Amoebiasis 8 1 i ; : : : Hydatid disease i 8: A Uninfected controls 10 0 Sensitivity (%) 4i 6i 90 4: 1: t: z :: ti 85 88 80 Specificity (%) 100 93 ‘Diagnosis based on clinical symptoms, microscopical examination of stool specimens and/or serological tests (cotmterimmunoelectrophoresis and indirect haemagglutination test). bathe cut-off limit for discriminating negative from positive results was determined by the mean value of the negative control +2 standard deviations. Table 2. Serodiagnosis of fascioliasis using crude F. gikantica III), and pooled antigen fractions 2 and 10

Total no. of patients

Clinical cases Fascioliasis s. nlansoni S. haenuatobium S. mansoni & S. haematobium

antigen, partially

purified antigen (peaks II and

Number of patients serologically”positive Crude antigen Peaks II & III Fractions 2 & 10 ELISA CIEP ELISA CIEP CIEP ELISA

20 f

20 2

6

2

20

20

z

8

Amoebiasis

20 1

8

2

: :1 A 8 ; Hydatid disease 10 Uninfected controls “CIEP=counter-immunoelectrophoresis;ELISA=enzyme-linked immunosorbentassay.

Acknowledgements

This work was supported by the Naval Medical Research and Development Command, Bethesda, Maryland 20814, Work Unit no. 3Ml62770A870.AQ.320. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the Navy Department, Department of Defense, or the US Government. References

Blagg, W., Schloegel, E. L., Mansour, N. S. & Khalif, G. I. (1955). A new concentration technique for demonstration of protozoan and helminth eggs in feces. American Journal

of Tropical

Medicine

and Hygkne,

4, 23-28.

Gottstein, B., Eckert, J. & Fey, H. (1983). Serological differentiation between Echinococcus granulosus and E.

20

8

8

8

z

8

8

z

:

would have similarpotential for specificserodiagnosis of fascioliasisor for non-specific crossreactivity. The ELISA using pooledfractions 2 and 10 of F. gigantica worm should be consideredas a rapid, highly sensitiveand specificserodiagnostictechnique for human fascioliasis.This test hasexcellent potential as an epidemiologicalsurveillancetechnique for determining prevalence of fascioliasisinfection in endemic areas.

20

in man. Zeitschrsft fiir Parasirenkunde, 69, 347-356. Hillyer, G. V. & Galanes, M. S. (1988). Identilication of a 17kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot te15iique. Journal of Clinical Microbiology, 26, 2048multilocularis

Laemmh, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Mansour, N. S., Youssef, F. G., Mikhail, E. M. & Boctor, F. N. (1983). Use of a partially purified Fasciola gantica worm antigen in the serological diagnosis oP human fascioliasis in Egypt. American Journal of Tropical Medicine and Hygiene, 32, 550-554. Santiago, N. & Hillyer, G. V. (1986). Isolation of potential serodiagnostic Fasciola hepatica antigens by electroelution from polyacrylamide gels. American 3oumul of Tropical Medicine and Hygiene, 35, 1210-1217. Watanabe, S. (1%5). A revision of the genusFasciola in Japan, with particular reference to F. hepatica and F. gigantica. In: Progress of Medical Parasiwlogy in Japan, vol. 2, Morishita, K., Komiya, Y. & Matsubayashieds, H. (editors). Tokyo: Megura Parasitological Museum, pp. 35s381. Received accepted

3 March 1990; revised publication 22 January

for

I.5 January 1991

1991;