Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti

Tropical Medicine and International Health

volume 3 no 1 pp 46–51 january 1998

© 1998 Blackwell Science Ltd46

Secretory acetylcholinesterase of Setaria cervi microfilariae andits antigenic cross-reactivity with Wuchereria bancrofti

S. Sharma, S. Misra and S. Rathaur

Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India

Summary Setaria cervi, a bovine filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant

amount of enzyme activity was detected both in culture media and somatic extracts of different

developmental stages of the parasite. The microfilarial stage showed a higher level of AChE activity than

adult worms, with females being considerably more active than males. The secretory enzyme from

microfilariae preferentially utilized acetylthiocholine iodide as substrate and showed two electrophoretically

distinct isoforms in native PAGE. Secretory enzyme was purified from the excretory/secretory products of

microfilariae using edrophonium chloride linked to epoxy-activated sepharose. Analysis of purified

acetylcholinesterase by SDS-PAGE revealed the existence of two proteins of 75kD and 45kD under non-

reducing conditions. These secretory enzymes are antigenic and cross-reactive with Wuchereria bancrofti-

infected asymptomatic microfilaraemic human sera when tested by enzyme linked immunosorbent assay and

immunoblotting. The secretory AChE(s) from S. cervi microfilariae may be utilized for diagnosis of early

filarial infections.

keywords AChE, S. cervi, W. bancrofti, diagnosis

correspondence Dr. S. Rathaur, Department of Biochemistry, Faculty of Science Banaras Hindu

University, Varanasi-221 005, India

Introduction

The secretion of acetylcholinesterase (AChE) by nematode

parasites is well established (Rhoads 1981). A number of

filarial parasites release AChE into the culture medium when

maintained in vitro (Rathaur et al. 1987, 1992b), representing

an active secretory process. Setaria cervi, a bovine filarial

parasite homologous with Wuchereria bancrofti (Kaushal et

al. 1987), releases a significant amount of AChE during in

vitro culture. However, the role of secretory AChE is still

speculative (Rhoads 1984, Lee 1996).

The success of serological diagnosis with

excretory/secretory (ES) antigens in several parasitic

infections, e.g. Toxocara canis (Hogarth-Scott 1966, Brunello

et al. 1986) prompted substantial recent interest in the use of

ES antigens for serological diagnosis of lymphatic filariasis

and onchocerciasis. Microfilarial, L3 or adult ES antigens have

been used in the diagnosis of bancroftian (Harinath et al.

1984, Malhotra & Harinath 1984) and brugian filariasis by

Kaushal et al. (1984). There have been several reports of

experiments detecting circulating antigens in filarial

infections using polyclonal (Hamilton et al. 1984) and

monoclonal antibodies (Dissanayke et al. 1984, Sutanto et al.

1985). Recently, circulating parasite AChE was detected in

human filariasis (Rathaur et al. 1992a, Misra et al. 1993).

Detection of antibodies against ES antigens, together with

identification of circulating parasite ES product, may in

combination provide the best serological test for diagnosis of

filarial infections.

We purified AChE from the microfilarial excretory/

secretory product and studied its cross-reactivity with

W. bancrofti infection. Secretory AChE from S. cervi

microfilariae (mf) cross-reacted with the W. bancrofti-

infected asymptomatic microfilaraemic human sera as studied

by ELISA and immunoblotting. We suggest that the detection

of AChE antibodies or circulating AChE using S. cervi AChE

antigen or antibody could be used for the diagnosis of early

filarial infection.

Materials and methods

Acetylthiocholine iodide (ATCH). butyrylthiocholine iodide

(BTCH). eserine. edrophonium chloride, epoxy-activated

sepharose 6B, anti-human immunoglobulin peroxidase

Tropical Medicine and International Health volume 3 no 1 pp 46–51 january 1998

S. Sharma et al. Antigenic cross-reactivity of Setaria cervi and Wuchereria bancrofti

© 1998 Blackwell Science Ltd 47

conjugate, orthophenylenediamine chromogen (OPD) and

diaminobenzidine hydrochloride (DAB) were purchased from

Sigma (St. Louis, MO, USA). All other chemicals used were of

analytical grade purity.

Adult S. cervi were recovered from the peritoneal folds of

freshly slaughtered water buffaloes. Worms were washed with

saline and stored in Krebs Ringer bicarbonate buffer

supplemented with 1% glucose, 100 mg/ml penicillin, 100

mg/ml streptomycin and 2mm fresh glutamine. Microfilariae

(mf) were obtained by dissecting the distal portion of the

uterus of adult females and incubated with supplemented

KRB buffer for 2 h at 37 8C under sterilized conditions for the

collection of ES products. For enzymatic studies, pooled ES

material was concentrated 4–5 times and dialysed against

0.1M phosphate buffer pH 7.6 at 4 8C. Worm extract was

prepared by homogenizing adult worms at 4 8C in PBS pH

7.6, centrifuging at 10,000 3 g and recovering the clear

supernatant. Microfilariae were sonicated using a MSE150W

ultrasonic disintegrator MK2 at 20 KC for 15 min with 2 min

interval after every 2 min in cold PBS pH 7.6. Protein

estimation was done by the method of Lowry (1951).

Acetylcholinesterase was assayed according to the method of

Ellman et al. (1961), with ATCH and BTCH as substrates.

The ES enzyme was resolved under non-denaturing

conditions by electrophoresis in 6% polyacrylamide gels in

Tris-borate EDTA (TBE) buffer pH 8.0 and enzyme activity

detected according to the direct colouring thiocholine method

of Karnovsky & Roots (1964).

W. bancrofti-infected human sera were collected from

Chiraigaon, an endemic area near Varanasi (India). Filarial

cases were divided into asymptomatic microfilaraemic (mf

carrier) and symptomatic amicrofilaraemia (elephantiasis).

Of the total population in Chiraigaon, 8% were found to be

asymptomatic microfilaraemic. Sera were collected from 25

such patients aged 20–45 years with 200–300 mf/mm3 of

blood. Healthy persons devoid of any infections living in the

endemic area were treated as endemic normal and those

living in a non-endemic area as non-endemic normal. Serum

was separated by keeping the blood at 4 8C for a few hours

and centrifuged at 10,000 rpm for 20 min in a refrigerated

centrifuge and stored at 270 8C till further use.

For the purification of acetylcholinesterase by affinity

chromatography, edrophonium sepharose was prepared

according to the method of Hodgson and Chubb (1983) and

washed in sequence with 10 volumes each of 0.1m sodium

acetate pH 4.5, 0.012 m sodium borate pH 10.0 and distilled

water. A 5ml column was prepared at 4 8C and the flow rate

adjusted to 12ml/h. Concentrated ES (5 mg) from S. cervi mf

was applied and the column sequentially washed with 50 mmphosphate buffer pH 8.0 and 50mm phosphate buffer

containing 0.5 m NaCl. Bound AChE was eluted with 50 mmphosphate buffer containing 0.5 m NaCl and 12 mmedrophonium chloride. Fractions of 2 ml were collected and

edrophonium removed by dialysis against 5 3 1 litre changes

of phosphate buffer pH 8.0 prior to the enzyme and protein

assays. Homogeneity of enzyme active peak fractions was

checked by 10% SDS-PAGE and silver staining of an aliquot

of the sample.

ELISA was performed following the procedure of Rathaur

et al. (1987). NUNC plates were coated with 2 mg/ml of

affinity purified enzyme and incubated overnight at 40 8C,

followed by the addition of 1:100 dilution of W. bancrofti-

infected asymptomatic microfilaraemic human sera as

antibody and anti-human IgG peroxidase conjugate (1:4000

dilution). Immunoblotting of secretory AChE was performed

according to Lunde et al. (1988) utilizing infected human sera

as antibody (1:20 dilution) and anti-human IgG peroxidase

conjugate (1:1000). Bands were stained by carrying out DAB

reaction.

Results and discussion

Our study demonstrates that both adult and microfilarial

stages of S. cervi contain acetylcholinesterase activity. Mf

secrete several forms of the enzyme actively when maintained

in vitro. Table 1 shows the specific activity of AChE in

extracts of adult worms, mf and ES product of the

microfilarial stage. On the basis of units/mg of protein, mf of

Enzyme activity Specific activity

Stage (units/ml) (units/mg)

Adult male 0.0275 6 0.005 0.0100 6 0.005

Adult female 0.1393 6 0.032 0.0366 6 0.0025

mf 1.4016 6 0.120 0.2922 6 0.021

ES product 0.2446 6 0.008 0.0466 6 0.001

The enzyme activity was measured according to the procedure of Ellman et al. (1961), using

ATI as substrate with 1 enzyme unit being expressed in mmoles of substrate hydrolysed per

minute. The data represent mean values 6 SD for 5 different determinations.

Table 1 AChE activity in adult worm

homogenate, microfilarial extract and

microfilarial ES product of S. cervi




S. cervi showed significantly higher activity than the adult

stage, with females being considerably more active than male

worms. The secretory AChE from mf preferentially utilized

ATCH over BTCH as substrate (data not shown). Greater

AChE activity per unit weight of protein in female worm

extracts than in males confirms the reported heterogeneity

between species (Rathaur et al. 1987).

We observed a considerable quantitative difference between

developmental stages, with microfilariae showing

approximately 8 times the activity of female adults. Similarly,

Hemonchus contortus larvae (Hart & Lee 1966), Brugia

malayi mf (Rathaur et al. 1987) and fourth-stage larvae of

Trichostrongylus colubriformis and Oesophagostomum

radiatum (Ogilvie et al. 1973) also show greater AChE

activity than adults. Lawrence & Pritchard (1993) found that

Heligmosomoides polygyrus secreted AChE with maximum

production of the enzyme occurring in the secretions from

the fourth-stage larvae. In contrast to this, there is marked

increase in enzyme activity of Nippostrongylus brasiliensis

during development.

The nematode secretory cholinesterases characterized so

far are either present as a single protein (Rothwell et al. 1973)

or in multiple molecular forms as in N. brasiliensis.

Blackburn & Selkirk (1992) showed that the

excretion/secretions of adult N. brasiliensis contain 3 major

forms of AChE and these were equivalent to forms A, B and

C identified by Edwards et al. (1971); 2 minor forms of AChE,

B1 and B2 were also sometimes present. McKeand et al.

(1994b) demonstrated that adults of the cattle lungworm

Dictyocaulus viviparus secrete 5 isoforms of the enzyme. In

our case, microfilarial secretory AChE resolved into two

distinct bands under 6% non-denaturing gel electrophoresis,

suggesting that S. cervi mf secrete at least two electromorphic

variants of AChE (data not shown).

The purification of AChE on edrophonium sepharose was

effected using the conditions described in Materials and

Methods with 53-fold enrichment of enzyme activity and

relatively low recovery of 9%. The loss in enzyme activity

may be attributed to dialysis during desalting of peak

fractions, as AChE is unstable to physical processes. The

purification of AChE from other parasites using this method

has been achieved with Necator americanus by Pritchard et

al. (1991) and T. colubriformis by Griffith & Pritchard

(1994).

Unfractionated microfilarial ES, detected using non-

reducing 10% SDS-PAGE on silver staining, shows several

bands ranging from 15 to 200 kD (Figure 1). The purified

enzyme from the edrophonium column was resolved only in

two proteins: a major component of 45kD and a very faint

band of 75 kD. Similar results have been found in other

invertebrates. Blackburn & Selkirk (1992) found isoforms of

AChE of 74 kD and 39 kD, both secreted by 4-day-old

worms, but there was a switch to predominant secretion of

the 39 kD form by day 8 of infection. They suggested that

this change may be related to maturation of the nematodes.

Nakazawa et al. (1995), with SDS-PAGE under non-reducing

conditions, detected only a 74 kD form of the enzyme in the

E/S of adult Nippostrongylus.

Cross-reactivity with heterologous antigens is common in

many tropical diseases (Kaliraj et al. 1979) but the degree of

specificity describes the suitability of such an antigen in

detecting the disease by immunological methods. Using

techniques of diagnostic potential, such as ELISA and

immunoblotting, circulating parasite antigens have been

detected in both human (Dasgupta et al. 1984, Hamilton et

al. 1984, Sugunan & Kaleysa Raj 1990) and animal

(Dasgupta et al. 1984) filariasis by polyclonal sera. In

humans, AChE activity has been found in immune complexes

obtained from filarial patients (Rathaur et al. 1992a).

Espinoza et al. (1988) reported antibodies against

Schistosoma AChE in the sera of mice and of human patients

infected with this parasite. McKeand et al. (1994a)

demonstrated that sera from calves infected naturally and

experimentally with D. viviparus contained antibodies which

Figure 1 Purification of S. cervi microfilarial secretory

acetylcholinesterase. Samples were run on 10% SDS-PAGE and silver-

stained. A, Fraction unabsorbed on edrophonium sepharose column;

B, crude ES originally loaded on column; C, AChE fraction eluted

with edrophonium chloride from same column.




specifically recognized secretory AChE of the adult parasite.

Secretory acetylcholinesterase purified from S. cervi

microfilarial ES, when reacted with W. bancrofti-infected

asymptomatic microfilaraemic human sera, showed

significantly higher activity in ELISA than endemic and non-

endemic normals (Figure 2). The mean OD of 10 non-

endemic normal sera evaluated was 0.127 (SD 5 0.017), so

the values above 0.161 were considered to have significantly

greater reactivity towards the secretory AChE in ELISA. The

mean OD for the endemic normal (n 5 10) was 0.183 (SD 5

0.010), which is slightly higher than normal. Significantly

higher reactivity was observed for human microfilaraemic

sera; the mean OD for 10 sera was 0.505 (SD 5 0.042). These

results clearly indicate that the AChE from S. cervi

microfilarial secretion is cross-reactive to W. bancrofti-

infected human sera and showed more than 3 times greater

activity than sera from non-filarial healthy humans. This

cross-reactivity of secretory AChE from S. cervi microfilariae

against sera from human filarial patients is in accord with the

view that homologous antigens are produced by related

species – as in this case between S. cervi and W. bancrofti.

Similar cross-reactivity has been shown by Sugunan and

Kaleysa Raj (1990) by S. digitata ES antigen.

Evidence for cross-reactivity was further provided by

immunoblotting. To check the cross-reactivity, we examined

the binding of W. bancrofti-infected human sera to the S.

cervi secretory AChE by immunoblotting. As shown in Figure

3, IgG antibodies in infected human sera recognized 45 kD

and 75 kD proteins specifically. Ott et al. (1975) and

Rosenberry (1977) reported that catalytic subunits of AChE

are typically around 75–80kD. Nakazawa et al. (1995)

reported that in immunoblotting analysis of ES product of N.

brasiliensis, a 74 kD AChE molecule is recognized by IgE and

IgG antibodies in nematode-infected rat sera. A report by

Blackburn & Selkirk (1992) showed that N. brasiliensis

secretory AChEs fall in two distinct proteins of 39 & 74 kD

with a high degree of structural identity, which appear to

derive from a single primary translation product. All AChEs

Figure 2 Reactivity of S. cervi secretory AChE in ELISA with

endemic normal (EN), non-endemic normal (NEN) sera and

W. bancrofti-infected asymptomatic microfilariaemic human sera

(Inf) from Chiraigaon (India). Horizontal bars represent mean OD

and dashed line indicate normal. Number of sera in each case was

10.

Figure 3 Immunoblot analysis of the affinity purified secretory

AChE of S. cervi mf. The purified enzyme was run on 10% SDS-

PAGE and transferred onto a nitrocellulose membrane. The

membrane was incubated with normal (Lane A) and W. bancrofti-

infected asymptomatic microfilaraemic human sera (Lane B). The

membrane was then incubated with anti-human IgG peroxidase

conjugate and the DAB reaction was carried out.



reported so far are heavily glycosylated and this could

account for the multiple isoforms seen under non-denaturing

PAGE, particularly as differing carbohydrate side-chains are

known to account for the 5 electrophoretically variant forms

of human enythrocyte AChE (Ott et al. 1975). Selkirk &

Blackburn (1992) proposed that resistance of native N.

brasiliensis AChE to trypsin digestion may be due to

extensive glycosylation.

Recognition of S. cervi secretory AChEs by W. bancrofti-

infected asymptomatic microfilaraemic human sera suggests

that clear cross-reaction between the two genera and the

recognition of this enzyme may be an interesting component

of the immune response in natural infection. Naturally

evoked serum antibodies to parasitic cholinesterase have also

been observed in animal infections with N. brasiliensis (Jones

& Ogilvie 1972), O. radiatum (Bremner et al. 1973),

Stephanurus dentatus (Massouli & Bon 1982) and in the

serum of infected humans. This cross-reactivity might be the

result of the presence of a common antigenic determinant

such as the carbohydrate moiety of these glycoprotein

enzymes or a common tertiary or quarternary structure of

the enzyme in the two species (Tarrab-Hazdai et al. 1984).

The conservation of AChE secretion among parasitic

helminths is indicative of an important role in the host-

parasite relationship. Lee (1996) proposed that these secretory

AChEs from parasitic nematodes may have an important role

in modulation of the host’s inflammatory and/or immune

response. Further experimental investigations are required for

assessing the role of S. cervi microfilarial secretory AChE in

the host-parasite relationship. In order to evaluate the

diagnostic potential of S. cervi secretory AChE under field

conditions in early filarial infections, work is in progress to

identify its amino-acid sequence, which could then be used to

produce recombinant protein.

Acknowledgements

S. Sharma is grateful to UGC (GATE) and S. Misra thanks

CSIR for providing financial assistance.

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Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti