diagnostic value of different antigenic fractions of hydatid cyst fluid from camel and sheep in kingdom of saudi arabia

ORIGINAL ARTICLEDiagnostic value of different antigenic fractions of hydatid cyst fluid from camel and sheep in Kingdom of Saudi Arabia Zoology Department, Faculty of Science, King Saud Un

ORIGINAL ARTICLE

Diagnostic value of different antigenic fractions of hydatid cyst fluid from camel and sheep in Kingdom of Saudi Arabia

Zoology Department, Faculty of Science, King Saud University, Saudi Arabia

Received 20 December 2010; accepted 2 January 2011

Available online 5 January 2011

KEYWORDS

Serodiagnosis;

Enzyme-linked

immuno-electrotransfer blot (EITB);

Hydatidosis;

Echinococcus granulosus;

KSA

Abstract Hydatid cyst fluids (HCF) crude extracts from camels and sheep slaughtered in Riyadh region, KSA were subjected to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS– PAGE) and Western blot analysis Sera from 17 confirmed human cases of hydatidosis, 25 patients with other parasitic infections and 10 clinically healthy subjects were used to evaluate the diagnostic value of the different antigenic fractions of these extracts Immunoblotting results revealed that, at least 11 major discrete protein fractions (110–8 kDa) were recognized by sera from hydatidosis patients, sera from patients with other parasitic diseases showed cross-reactivity with few of these bands The cluster of bands (38–35 kDa) that may be a breakdown of ‘‘Arc 5’’ antigen (39–38 kDa) was detected by 100% and 94% of sera from hydatidosis cases with HCF extracts from camel and sheep, respectively This cluster showed also some cross reactivity (20% and 8%) with control sera from patients with other parasitic infections with camel and sheep HCF extracts, respectively Poly-peptides at 24–22, 16 and 8 kDa which may probably correspond to antigen B subunits were also identified by all samples from hydatidosis patients with sheep HCF extracts and by 100%, 65% and 74% with camel HCF extracts respectively Sera from control subjects did not react with any of

* Corresponding author Address: Women Students, Medical

Stud-ies & Sciences Sections, King Saud University, P.O Box 22452,

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E-mail addresses: eolayan@hotmail.com (E.M Al-Olayan), helmy26_

2000@yahoo.com (H Helmy).

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these polypeptides (24–22, 16 and 8 kDa) According to our results, the identified molecular weight bands (16 and 8 kDa using HCF crude extracts from sheep and 24–22 kDa using HCF crude extracts either from camel or sheep) represent good candidates for immunodiagnosis of hydatidosis

ª 2011 King Saud University Production and hosting by Elsevier B.V All rights reserved.

1 Introduction

Hydatidosis is a disease caused by infection with the

metaces-tode stage of the dog tapeworm, Echinococcus granulosus The

disease represents a significant public health problem in several

North Africa and Middle East countries (Battelli et al., 2002;

Kamhawi, 1995; Sadjjadi, 2006; Shambesh et al., 1997) Several

studies indicated that hydatid disease is an endemic zoonosis in

the Kingdom of Saudi Arabia (KSA) affecting both humans

and their domestic animals (Abu-Eshy, 1998; Adewunmi and

Basilingappa, 2004; Al Mofleh et al., 2000; Fahim and Al

Sal-amah, 2007; Ibrahim, 2010; Rashed et al., 2004)

There is usually no direct parasitological evidence for the

presence of cysts in organs or tissues and in most cases the

early stages of infection are asymptomatic Over the last

dec-ade diagnosis of hydatid disease was improved due to the

use of imaging techniques including ultrasonography,

com-puted tomography (CT scanning) and magnetic resonance

imaging (MRI) supported by immunological assays for

confir-mation of clinical diagnosis (World Health Organization,

2003; Zhang et al., 2003)

Hydatid cysts of E granulosus develop in internal organs

(mainly the liver and lungs) of humans and intermediate hosts

(herbivores including: sheep, horses, cattle, pigs, goats and

camels) as unilocular fluid-filled bladders (Zhang et al.,

2003) Most studies have focused on hydatid cyst fluid

(HCF) antigens as an invaluable source of antigenic material

for immunodiagnosis (Burgu et al., 2000; Kanwar et al.,

1994; Musiani et al., 1978; Oriol et al., 1971; Piantelli et al.,

1977; Pozzuoli et al., 1974) Detection of antibodies to the well

characterized lipoproteins antigen B (AgB) and antigen 5 ‘‘Arc

5’’ (Lightowlers et al., 1989; Oriol and Oriol, 1975; Verastegui

et al., 1992), the major antigenic components of HCF, have

re-ceived most of the attention for such diagnostic approach

Although AgB and ‘‘Arc 5’’ have proved to be diagnostically

valuable, there are difficulties related to their lack of sensitivity

and specificity and problems with the standardization of their

use (Babba et al., 1994) Nevertheless, several studies evaluated

the immunogenicity of crude or semi-purified HCF antigens

derived from sheep (Rogan et al., 1991) or camel hosts (Ramzy

et al., 1999; Shambesh et al., 1995) with sera from patients with

hydatidosis which resulted in the development of promising

diagnostic systems However, there are considerable

differ-ences among the various tests in both specificity and

sensitiv-ity Enzyme-linked immunoelectrotransfer blot (EITB) was

reported to be the most sensitive serological assay for

confir-mation of hydatidosis (Verastegui et al., 1992) It also showed

high specificity due to the high resolution of HCF antigenic

components (Kharebov et al., 1997)

It is generally accepted that parasites accommodated in

different hosts or collected from a variety of geographical

localities would be of distinguished strains and even considered

as different species (Thompson and McManus, 2002), likely to

produce diverse antigenic constituents This may explain why

different laboratories using antigens prepared from

different host species obtain different anti-hydatid antibody relativities

The present study was designed to evaluate and compare the diagnostic value of different antigenic fractions of HCF crude extracts from camel and sheep origin using EITB technique

2 Materials and methods 2.1 Antigens preparation Fertile hydatid cysts were obtained from six sheep and three camels within 24 h of slaughter in the main abattoir in Riyadh region, Kingdom of Saudi Arabia A single lot of crude anti-gen extract was prepared from each animal source according

toRamzy et al (1999)with slight modifications Briefly, hyda-tid cyst fluid (HCF) was aspirated aseptically and 25· protease inhibitor cocktail (Roche, Basel, Switzerland) in 100 mM phosphate buffer, pH 7.0 was immediately added HCF was centrifuged at 3000g for 30 min at 4C and the supernatant was collected and dialyzed, using cellulose membrane with molecular weight cut-off of 3.5 kDa, against three changes of deionized water per 24 h over three successive days The dial-yzate was centrifuged again at 3000g for 30 min at 4C and the supernatant collected Protein content was estimated by the Bio-Rad Bradford protein assay kit (Bio-Rad AG, Glattb-rugg, Switzerland) using bovine plasma gamma globulin as a standard The two prepared antigen extracts were divided into aliquots and stored at 70C until used

2.2 Serum samples Serum samples for the present study were collected from 17 surgically confirmed human cases with hydatidosis (group A) Control sera were obtained from 25 patients with parasitic infections other than E granulosus (group B), and from 10 clinically healthy subjects with no history of living in endemic areas and free from parasitic infections (group C) Group B, consisted of 4 cases with amoebic liver abscesses, 3 cases with ascariasis, 3 cases with schistosoiasis, 5 cases with fascioliasis,

5 cases with cysticercosis and 5 cases of ancylostomiasis 2.3 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Enzyme-linked immunoelectrotransfer blot (EITB)

HCF extracted antigens from sheep and camel were analyzed basically as described byLaemmli (1970) Briefly, 100 mg from each source were separated by SDS–PAGE under denaturing conditions using 5–20% gradient gels Fractionated proteins were electrophoretically transferred to nitrocellulose mem-branes as described byVerastegui et al (1992) After blotting, nitrocellulose sheets were cut into 0.3 cm strips, blocked in 0.01 M phosphate buffered saline (pH 7.4) with 0.05% Tween

20 (PBS/T) and 5% non fat dry milk for 30 min, and incubated

with human sera (1:250 in PBS/T with 5% fetal calf serum

[FCS]) for 2 h at 37C After washing the strips were

incu-bated in alkaline phosphatase conjugated antihuman IgG

anti-bodies (Promega, Madison, WI, USA) in PBS/T with 5% FCS

for 2 h at 37C After washing the nitrocellulose strips were

developed with the substrate 5-bromo-4-chloro-3-indolyl

phos-phate/nitroblue tetrazolium (BCIP/NBT, Sigma Chemical Co.,

St Louis, MO, USA) in 100 mM Tris–HCl with 100 mM NaCl

and 5 mM MgCl2for 15 min at 37C

Molecular weight estimates were made by comparing the

mobility of the different antigen fractions with these of

Pre-stained Protein Marker, Broad Range (7–175 kDa), (New

Eng-land Biolabs, USA) which were separated in the same gel of a

hydatid fluid sample and electrotransferred to the same sheet

3 Results

Sera of the 17 confirmed hydatidosis cases (group A)

recog-nized at least 11 major bands in HCF antigens originated from

camel and sheep cysts (Fig 1) with molecular weights of (110,

97, 80, 65–60, 55–50, 45–40, 38–35, 30, 24–22, 16.8 kDa) Some

sera from patients with other parasitic diseases (group B) showed cross-reactivity with few numbers of these bands Sera from clinically healthy individuals (group C) did not show any reactivity to these polypeptides.Fig 2, represents the differ-ences in the reactivity to these antigenic fractions from camel and sheep HCF extracts with the studied sera groups The cluster of bands at 45–40 kDa was detected by 94% and 77% of hydatidosis cases with HCF extracts from camel and sheep respectively Though, this cluster of bands (45–

40 kDa) was not detected by any of the controls with HCF ca-mel extracts, it was detected by 16% of control sera from group B with HCF sheep extracts (2 cases of ancylostomiasis and 2 cases of fascioliasis)

While, the cluster of band at 38–35 kDa was detected by all hydatidosis sera samples with HCF camel extracts and by 94% with HCF sheep extracts This cluster showed also some cross reactivity (20% and 8%) with control sera from group B with camel and sheep HCF extracts respectively (3 cases of cysticer-cosis and 2 cases of amoebic liver abscesses with HCF camel extracts and 2 cases of cysticercosis with HCF sheep extracts) The cluster of bands at 24–22 kDa was found to be com-mon (100%) in all hydatidosis cases using camel and sheep

Figure 1 Immunoblot analysis of HCF preparations from camels and sheep showing the different recognized antigen fractions by the studied groups, each strip shows the IgG antibody binding of a single serum sample Approximate molecular weight (MW) markers are indicated (A) 17 cystic hydatidosis patients; (B) 25 patients with other parasitic infections: 1–4 amoebic liver abscess, 5–7 ascariasis, 8–10 schistosomiasis, 11–15 fascioliasis, 16–20 cysticercosis, 21–25 ankylostomiasis; (C) 10 clinically healthy individuals

HCF extracts Moreover, sera from patients with other

para-sitic diseases (group B) and from normal controls (group C)

did not react with any of these polypeptides

Though, the relatively low molecular weight of bands at 16

and 8 kDa were detected by 100% of sera from hydatidosis

cases with sheep HCF extracts; they were detected by 65%

and 74% of CE cases with camel HCF extracts, respectively

These bands (16 and 8 kDa) showed 100% specificity using

both antigen extracts

4 Discussion

The present work is one of the first studies in Saudi Arabia to

analyze protein extracts of HCF collected from infected camels

and sheep using SDS–PAGE and EITB techniques to identify

their antigenic fractions and compare the diagnostic values of

these fractions Sera samples from surgically confirmed

hyda-tidosis cases were used for identifying antigenic determinants

in HCF which are unshared with control sera samples (patients

with other parasitic infections and clinically healthy

individuals)

Immunoblotting results identified 11 major discrete

anti-genic fractions (110–8 kDa) in HCF preparations from camel

and sheep Our data are in agreement with those obtained by

another group (Kanwar et al., 1992); they reported that sera

from surgically confirmed cases of hydatidosis reacted with

12 polypeptides with molecular weights of 8–116 kDa by

Wes-tern blotting using hydatid antigens from sheep

In the present study, sera of the studied groups showed

some variations in recognizing the 11 detected protein

frac-tions (110–8 kDa) between HCF preparafrac-tions from camel

and sheep Though, the cluster of bands at 45–40 kDa was

one of the most frequently recognized bands by CE cases (94%), this in addition to absolute specificity with HCF camel extracts It was detected by only 77% sensitivity and 84% specificity with HCF sheep extracts

It is interesting to note that, the cluster of bands (38–

35 kDa) that may be a breakdown of ‘‘Arc 5’’ antigen (39–

38 kDa) (Shepherd and McManus, 1987) was detected in the present study by 100% and 94% of sera from CE cases with HCF extracts from camel and sheep, respectively Detection

of antigen ‘‘Arc 5’’ is one of the most widely used immunodi-agnostic procedures for hydatid disease (Zhang et al., 2003)

On the other hand, a relatively low specificity (80% and 92%) was recognized towards these polypeptides with camel and sheep HCF extracts respectively This coincides with many studies that reported cross-reactivity of antigen ‘‘Arc 5’’ (39–

38 kDa subunits) with sera from patients with other parasites (Lightowlers and Gottstein, 1995; Liu et al., 1993; Ortona et al., 2000; Poretti et al., 1999; Rott et al., 2000) as a major prob-lem The cross reactivity of this antigen may be attributed to its phosphorylcholine epitope, first described by Shepherd and

McManus (1987) and later confirmed by Lightowlers et al

(1989)

Furthermore, the immunogenic polypeptides at 24–22, 16 and 8 kDa which may probably correspond to antigen B sub-units were also seen in the current study by all sera samples from CE patients with sheep HCF extracts and by 100%, 65% and 74% with camel HCF extracts respectively These polypeptides (24–22, 16 and 8 kDa) showed by 100% specific-ity, as sera from 25 patients with other parasitic diseases and from 10 clinically healthy individuals did not react with any

of these polypeptides with antigen extracts from both sources Antigen B is highly immunogenic and was described in previ-ous reports under reduced condition on SDS–PAGE, with three bands with molecular sizes of approximately 8 or 12,

16, and 24 kDa (Leggatt et al., 1992; Lightowlers et al.,

1989; Oriol et al., 1971) Other researchers reported also that, the smallest subunit of antigen B (8 kDa) is highly sensitive and is more specific than the others (Ali-Khan, 1974; Maddi-son et al., 1989)

Hence, generally, the smallest subunit has proved the most useful target in diagnostic studies (Ortona et al., 2000; Rott et al., 2000) Some of the antigenic bands detected in the present study by EITB (16 and 8 kDa using HCF crude extracts from sheep and 24–22 kDa using HCF crude extracts either from ca-mel or sheep) might be used as a confirmatory test in suspected cases of human infection with hydatid disease

In conclusion, this study has provided interesting data on the use of EITB as a powerful tool in the context of serodiag-nosis of human hydatidosis

References

Abu-Eshy, S.A., 1998 Some rare presentations of hydatid cyst (Echinococcus granulosus) J R Coll Surg Edinb 43 (5), 347–352 Adewunmi, O.A., Basilingappa, H.M., 2004 Primary ovarian hydatid disease in the Kingdom of Saudi Arabia Saudi Med J 25 (11), 1697–1700.

Al Mofleh, I.A., Al Rashed, R.S., Ayoola, E.A., Al Faleh, F.Z., Al Amri, S.M., Al Rikabi, A.C., Al Sohaibani, M.O., Reyes, A.H.,

2000 Hepatic granulomas in an Arab population: a retrospective study from a teaching hospital in Riyadh Saudi J Gastroenterol 6, 41–46.

Figure 2 Frequency (% positive) of recognition of the major

HCF polypeptides in camels and sheep hosts by human sera

obtained from hydatidosis patients and controls (patients with

other parasitic diseases and clinically healthy individuals) as

characterized by their molecular weight

Ali-Khan, Z., 1974 Host-parasite relationship in Echinococcosis II.

Cyst weight, hematologic alterations, and gross changes in the

spleen and lymph nodes of C57L mice against graded doses of

Echinococcus multilocularis cysts J Parasitol 60, 236–242.

Babba, H., Messedi, A., Masmoudi, S., Zribi, M., Grillot, R.,

Ambriose-Thomas, P., Beyrouti, I., Sahnoun, Y., 1994 Diagnosis

of human hydatidosis: comparison between imagery and six

serologic techniques Am J Trop Med Hyg 50, 64–68.

Battelli, G., Mantovani, A., Seimenis, A., 2002 Cystic Echinococcosis

and the mediterranean region: a long-lasting association

Parassit-ologia 44, 43–57.

Burgu, A., Douanay, A., Gonenc, B., Sarimehmetoulu, H.O.,

Kalin-bacak, F., 2000 Analysis of fluids of hydatid cysts from sheep by

SDS–PAGE, and determination of specific antigens in protein

structure by Western blotting Turk J Vet Anim Sci 24, 493–500.

Fahim, F., Al Salamah, S.M., 2007 Cystic Echinococcosis in Central

Saudi Arabia: a 5-year experience Turk J Gastroenterol 18 (1),

22–27.

Ibrahim, M.M., 2010 Study of cystic Echinococcosis in slaughtered

animals in Al Baha region, Saudi Arabia: interaction between some

biotic and abiotic factors Acta Trop 113 (1), 26–33.

Kamhawi, S., 1995 A retrospective study of human cystic

Echinococ-cosis in Jordan Ann Trop Med Parasitol 89, 409–414.

Kanwar, J.R., Kaushik, S.P., Sawhney, I.M.S., Kamboj, M.S.,

Mehtas, S.K., Vnayak, V.K., 1992 Specific antibodies in serum

of patients with hydatidosis recognised by immunoblotting J Med.

Microbiol (36), 46–51.

Kanwar, J.R., Kanwar, R.K., Grewal, A.S., Vinayak, V.K., 1994.

Significance of detection of immune-complexed 8 kDa

hydatid-specific antigen for immunodiagnosis of hydatidosis FEMS

Immunol Med Microbiol 9, 231–236.

Kharebov, A., Nahmias, J., El-On, J., 1997 Cellular and humoral

immune responses of hydatidosis patients to Echinococcosis

granulosus purified antigen Am.J Trop Med Hyg 57 (5), 619–

625.

Laemmli, U.K., 1970 Cleavage of structural proteins during the

assembly of the head of bacteriophage T4 Nature 227,

680–685.

Leggatt, G.R., Yang, W., McManus, D.P., 1992 Serological

evalua-tion of the 12 kDa subunit of antigen B in Echinococcus granulosus

cyst fluid by immunoblot analysis Trans R Soc Trop Med Hyg.

86, 189–192.

Lightowlers, M.W., Gottstein, B., 1995 Echinococcosis/hydatidosis:

antigens, immunological and molecular diagnosis In: Thompson,

R.C.A., Lymbery, A.J (Eds.), The biology of Echinococcus and

hydatid disease CAB International, Wallingford, United

King-dom, pp 355–410.

Lightowlers, M.W., Liu, D.Y., Haralambous, A., Rickard, M.D.,

1989 Subunit composition and specificity of the major cyst fluid

antigens of Echinococcus granulosus Mol Biochem Parasitol 37,

171–182.

Liu, D., Rickard, M.D., Lightowlers, M.W., 1993 Assessment of

monoclonal antibodies to Echinococcus granulosus antigen 5 and

antigen B for detection of human hydatid circulating antigens.

Parasitology 106, 75–81.

Maddison, S.E., Slemenda, S.B., Schantz, P.M., Fried, J.A., Wilson,

M., Tsang, V., 1989 A specific diagnostic antigen of Echinococcus

granulosus with an apparent molecular weight of 8 kDa Am J.

Med Hyg 40, 377–383.

Musiani, P., Piantelli, M., Lauriola, L., Arru, E., Pozzuoli, R., 1978.

Echinococcus granulosus: specific quantification of the two most

immunoreactive antigens in hydatid fluids J Clin Pathol 31, 475–

478.

Oriol, C., Oriol, R., 1975 Physicochemical properties of a lipoprotein antigen of Echinococcus granulosus Am J Trop Med Hyg 24, 96–100.

Oriol, R., Williams, J.F., Esandi, M.V.P., Oriol, C., 1971 Purification

of lipoprotein antigens of Echinococcus granulosus from sheep hydatid fluid Am J Trop Med Hyg 20, 569–574.

Ortona, E., Rigano, R., Margutti, P., Notargiacomo, S., Ioppolo, S., Vaccari, S., Barca, S., Buttari, B., Profumo, E., Teggi, A., Siracusano, A., 2000 Native and recombinant antigens in the immunodiagnosis of human cystic Echinococcosis Parasite Immu-nol 22, 553–559.

Piantelli, M., Pozzuoli, R., Arru, E., Musiani, P., 1977 Echinococcus granulosus: identification of subunits of the major antigens J Immunol 119, 1382–1386.

Poretti, D., Felleisen, E., Grimm, F., Pfister, Teuscher., Zuercher, M.F.C., Reichen, J., Gottstein, B., 1999 Differential Immunodi-agnosis between cystic hydatid disease and other cross-reactive pathologies Am J Trop Med Hyg 60, 193–198.

Pozzuoli, R., Musiani, P., Arru, E., Patrono, C., Piantelli, M., 1974 Echinococcus granulosus: evaluation of purified antigens immuno-reactivity Exp Parasitol 35, 52–60.

Ramzy, R.M.R., Helmy, H., El Zayyat, E.A., Rifaat, M.M.A., Abdel Hameed, D.M., Abdel-Baki, M.H., 1999 An enzyme-linked immunosorbent assay for detection of IgG1 antibodies specific to human cystic Echinococcosis in Egypt Trop Med Int Health 4, 616–620.

Rashed, A.A., Omer, H.M., Fouad, M.A., Al Shareef, A.M., 2004 The effect of severe cystic hydatidosis on the liver of a Najdi sheep with special reference to the cyst histology and histochemistry J Egypt Soc Parasitol 34 (1), 297–304.

Rogan, M.T., Craig, P.S., Zeyhle, E., Romig, T., Lubano, G.M., Deshan, L., 1991 Evaluation of a rapid dot-ELISA as a field test for the diagnosis of cystic hydatid disease Trans R Soc Trop Med Hyg 85, 773–777.

Rott, M.B., Fernandez, V., Farias, S., Ceni, J., Ferreira, H.B., Haag, K.L., Zaha, A., 2000 Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation Acta Trop 75, 331–340.

Sadjjadi, S.M., 2006 Present situation of Echinococcosis in the Middle East and Arabic North Africa Parasitol Int 55, S197–S202 Shambesh, M.K., Craig, P.S., Gusbi, A.M., Echtuish, E.F., Wen, H.,

1995 Immunoblot evaluation of the 100 and 130 kDa antigens in camel hydatid cyst fluid for the serodiagnosis of human cystic Echinococcosis in Libya Trans R Soc Trop Med Hyg 89, 276– 279.

Shambesh, M.K., Craig, P.S., Ibrahem, M.M., Gusbi, A.M., Echtuish, E.F., 1997 A high prevalence of cystic hydatid disease in North Africa Ann Trop Med Parasitol 91, 957–959.

Shepherd, J.C., McManus, D.P., 1987 Specific and cross-reactive antigens of Echinococcus granulosus hydatid cyst fluid Mol Biochem Parasitol 25, 143–154.

Thompson, R.C.A., McManus, D.P., 2002 Towards a taxonomic revision of the genus Echinococcus Trends Parasitol 18, 452–457 Verastegui, M., Moro, P., Guevara, A., Rodriguez, T., Miranda, E., Gilman, R.H., 1992 Enzyme-linked immunoelectrotransfer blot test for diagnosis of human hydatid disease J Clin Microbiol 30, 1557–1561.

WHO Informal Working Group, 2003 International classification of ultrasound images in cystic Echinococcosis for application in clinical and field epidemiological settings Acta Trop 85, 253–261 Zhang, W., Li, J., McManus, D.P., 2003 Concepts in immunology and diagnosis of hydatid disease Clin Microbiol Rev., 18–36.