Technical Note

Antigenic response of midgut (Boophilus microplus)

cell culture vaccine

Masood Akhtar*, C.S. Hayat, Zafar Iqbal

Department of Veterinary Parasitology, University of Agriculture, Faisalabad 38040, Pakistan

Accepted 4 January 1999

Abstract

Antigenic response of midgut, Boophilus microplus, cell culture vaccine was evaluated. The antibody titres against the gut-

membrane (GM) and gut-soluble (GS) antigens were measured by indirect hemagglutination test. The antibody response

against GM antigen was signi®cantly higher (p < 0.01) than that of GS antigen. It was assumed on the basis of antibody titres

against GM and GS antigens that the in vitro propagated midgut cells, Boophilus microplus, contained substantial membrane

and membrane associated immunogenic components. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: Boophilus microplus; Cell culture; Gut soluble; Gut membrane; Antigenic response

1. Introduction

The ability to manipulate organisms on the mole-

cular level and the advent of modern immunological

procedures have provided potentially new alternatives

for tick control. Antigens from partially engorged

female Boophilus microplus have been used with

variable success to immunize animals against tick

infestation (Johnston et al., 1986). Midgut of the ticks

has been used as an immunogen (Opdebeeck et al.,

1988b, a; Jackson and Opdebeeck, 1990), but its

feasibility as a vaccine requires the production of

the antigenic material in large quantity. Partial success

with in vitro propagation of the midgut cells (Akhtar et

al., 1992) has encouraged the investigators to produce

a cell culture derived antitick vaccine. This paper

describes the immunogenic response to vaccine

prepared from in vitro propagated midgut cells of

Boophilus microplus.

2. Materials and methods

2.1. Chemosterilization of ticks

Fifty, semi-engorged, adult female, Boophilus

microplus, were obtained from the tick colony (Akhtar

and Hayat, 1993) maintained by the Department of

Veterinary Parasitology, University of Agriculture,

Faisalabad, Pakistan. Ticks were surface sterilized

by submersion and intermittent agitation in 0.5%

benzalkonium chloride solution. The ticks were then

washed with 70% alcohol until they were cleared of

Small Ruminant Research 33 (1999) 189±192

*Corresponding author.

0921-4488/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 0 1 1 - 5

benzalkonium chloride, and then rinsed in three

changes of sterilized distilled water.

2.2. Tissue extirpation and disaggregation

Chemosterilized ticks were dissected to separate

the midguts (Opdebeeck et al., 1988b). The dissected

guts were given several washings with balanced

salt solution (0.5 g/ml) and dissociated by a simple

teasing method (Guru et al., 1976). The suspension

was centrifuged at 1500 rpm at 48C for 20 min. The

sediment was washed twice with balanced salt solu-

tions and the cell pellet was collected for further

use.

2.3. In vitro propagation of midgut cells

Midgut cells of Boophilus microplus were propa-

gated on Grace's insect medium (Sigma, USA) sup-

plemented with 5% bovine foetal calf serum (heat

inactivated at 568C for 30 min) and antibiotics (Potas-

sium Penicillin 100 IU/ml and Streptomycin sulphate

100 mg/ml; Shanghai Pharmaceutical Industry, China)

along with fungizone (50 IU/ml) as described pre-

viously (Akhtar et al., 1992).

2.4. Cell harvesting

Cells adherent to the surface of culture ¯asks were

harvested physically using rubber policemen. Cell

culture suspension was removed and poured into

sterile centrifuge tubes. The ¯asks were rinsed 2±3

times with sterile phosphate buffered saline (PBS; pH

7.2) and added to the tubes containing cell culture

suspension. Finally, the pooled cell suspension was

centrifuged at 1500 rpm for 30 min at 48C. Super-

natant was discarded and sediment resuspended in

fresh PBS. The cells were given three washings with

PBS by centrifugation at 48C. Pellet was collected and

reconstituted in minimum volume of PBS. Protein

concentrations were assayed using the method of

Bradford (1976) and the suspension was diluted with

PBS to attain the desired concentration of 5 mg per ml.

2.5. Cell culture vaccine and its administration

Midgut cell culture vaccine was prepared by emul-

sifying the harvested cells in a double volume of

Freund's complete adjuvant (Sigma, USA). Twenty

male lohi sheep (2 years age, 30 kg average weight)

maintained by the Department of Veterinary Parasi-

tology, University of Agriculture, Faisalabad, Pakistan

were used as experimental model because sheep are

inexpensive and closely related immunologically to

cattle/buffalo (Tizzard, 1992). All the sheep were

rendered free from helminth parasites by using oxfen-

dazole, at 7 ml per animal orally Systamex ICI Pak.).

The parasite free status of the animals was con®rmed

through regular faecal examination for 2 weeks before

experiment. Animals were divided into two equal

groups. One group of 10 animals was inoculated with

midgut cell culture vaccine (2.5 ml/animal) intramus-

cularly followed by same booster dose 3 weeks later.

The 10 animals in the control group were injected with

adjuvant and PBS at the same time as the vaccinated

animals.

2.6. Antigens preparation for indirect

hemagglutination test

Midguts of the chemosterilized ticks, Boophilus

microplus, were dissected and gut-soluble (GS) and

gut-membrane (GM) antigens were prepared (Opde-

beeck et al., 1988b). Brie¯y, the guts were disrupted in

an ultra homogenizer (Ultra-Turrax, Janke and Kunkel

KG, Staufen, UK) in 0.15 M PBS, pH 7.2, containing

1 mM disodium EDTA, sonicated (Rapid 600, Ultra-

Sonic; Shipley Yorks, UK) for 10 min in 30±60 s

burst, and centrifuged at 5000 rpm for 1 h to yield

GS and membrane associated (GM) gut antigens. GM

was homogenized in 10 mM PBS, pH 7.2, containing

5 mM disodium EDTA. All procedures were carried

out at 4±88C.

2.7. Serology

Sheep were bled from the jugular vein pre-vaccina-

tion and at 21 days post-vaccination. Blood thus

collected was allowed to coagulate, the clot was

broken with a glass rod and blood tubes were kept

at 48C for over night. Serum then was aspirated with

Pasteur's pipettes and stored at ÿ208C till used. Sera

collected were tested for antibody levels by indirect

hemagglutination test (Akhtar, 1995) in comparison

with the positive and negative controls.

190 M. Akhtar et al. / Small Ruminant Research 33 (1999) 189±192

3. Results

Indirect hemagglutination (IHA) test was used to

detect the antibody titres in sera from sheep vaccinated

with midgut, Boophilus microplus, cell culture vac-

cine. Antitick antibody titres in pre-vaccinated sera

were nil in all the animals. The antibody titres against

GM an GS antigens were found in sera from all

vaccinated sheep but not in sera from control sheep

injected with adjuvant and PBS, 21 days post-inocu-

lation. The antibody response against GM antigen was

signi®cantly higher (p < 0.01) than that against GS

antigen (Table 1). The IHA antibody titre against GM

antigen ranged from 1 : 16 to 1 : 128. Among 10

samples processed, three each showed antibody titre

1 : 16 and 1 : 64 while two each showed antibody titre

1 : 32 and 1 : 128. On the other hand, the IHA anti-

body titre against GS antigen ranged from 1 : 2 to

1 : 32. Among 10 samples processed, the antibody

titres were 1 : 2, 1 : 4, 1 : 8, 1 : 16 and 1 : 32 in three,

one, three, one and two samples, respectively.

4. Discussion

Antibodies reactive with tick extracts have been

detected by a number of investigators (Willadsen,

1980; Wikel, 1984; Johnston et al., 1986; Opdebeeck

et al., 1988b; Jackson and Opdebeeck, 1989) but this is

the ®rst report which describes the immunogenic

activity of the midgut antigens derived from cell

culture. Moreover, IHA has been used for the ®rst

time for assaying the anti-tick antibody titres. The

antibody response against GM antigen was signi®-

cantly higher (p < 0.01) than that against GS antigen

(Table 1). Similar immune response against GM anti-

gen has been observed by Jackson and Opdebeeck

(1989) who have used midgut extract of Boophilus

microplus as vaccine in cattle. It is speculated that gut

membrane may contain B-cell mitogens which

potentiate synthesis of antibodies to the GM antigens

in the sheep vaccinated with midgut extract as in

case of cattle (Jackson and Opdebeeck, 1989). This

speculation can also be supported by the fact that

polyclonal activation of B-cells and (hypergamma

globulinaemia) are also associated with other parasitic

diseases, like malaria and trypanosomiasis (Wakelin,

1984).

Unfortunately challenge experiments to measure

protective immunity were not possible on sheep as

they are not regarded as suitable hosts for Boophilus

microplus because few ticks survive the entire life

cycle on sheep. But such a high level of antibody titres

have been correlated to protection following vaccina-

tion having GM components (Wong and Opdebeeck,

1989; Jackson and Opdebeeck, 1989). It can be

assumed on the basis of antibody titres against GM

and GS antigens that the in vitro propagated midgut

cells of the tick Boophilus microplus, contained con-

siderable membrane and membrane associated com-

ponents.

The use of parasite cell membranes as targets for

protective immunity has been reviewed by Gitter

(1983). Enzymes and chemoreceptors have been con-

sidered as membrane antigens and may form immu-

nological reactions which destroy the cell. Membrane

glycoproteins of parasites have been used for protect-

ing animals (Scott and Snary, 1979; Scott et al., 1985).

Johnston et al. (1986) protected cattle against Boo-

philus microplus with a crude adult tick extract which

included both particulate and soluble components or

particulate components only. Supernatant and pellet

prepared by centrifugation gave 79% and 65% protec-

tion, respectively against Boophilus microplus. Opde-

beeck et al., 1988b, a) have also reported high

protection (91%) by membrane antigen from the

midgut of Boophilus microplus challenge larval and

adult ticks.

Table 1

Antibody titre against GM and GS antigens of Boophilus microplus

in sheep vaccinated with cell culture vaccine at 21 days post-

vaccination. All titres were negative at day zero

Animals No. IHA antibody titre

GM GS

1 1 : 64 1 : 8

2 1 : 64 1 : 8

3 1 : 32 1 : 8

4 1 : 128 1 : 32

5 1 : 32 1 : 4

6 1 : 16 1 : 2

7 1 : 16 1 : 2

8 1 : 64 1 : 16

9 1 : 128 1 : 32

10 1 : 16 1 : 2

M. Akhtar et al. / Small Ruminant Research 33 (1999) 189±192 191

Better understanding of parasite cell membrane

structure would be of great help in selecting immuno-

gens. Membranes form a three dimensional ¯uid

mosaic in which proteins (including glycoproteins

and lipoproteins) are dispersed through a lipid layer

(Capaldi and Green, 1972; Singer and Nicolson,

1992). Most membrane proteins are organized into

complexes with molecular weights between 10 000

and 200 000 and subunits with molecular weights of

about 10 000±50 000 (Capaldi and Green, 1972).

Work is in progress to purify the cell membrane,

membrane-associated and subunit components from

the in vitro propagated midgut cells of the tick,

Boophilus microplus. Hopefully this will be helpful

in better vaccine preparation which would be more

effective in promoting protection than the midgut cell

culture vaccine used in the present study.

References

Akhtar, M., 1995. Studies on the immunization of buffalo against

Boophilus microplus. Ph.D thesis, University of Agriculture,

Pakistan.

Akhtar, M., Hayat, C.S., 1993. Ecology and laboratory propagation

of the tick Boophilus microplus. J. Anim. Plant Sci. 3, 78.

Akhtar, M., Hayat, C.S., Ashfaque, M., Iqbal, Z., Afaq, M., 1992.

In vitro propagation of midgut cell of Boophilus microplus.

Pakistan Vet. J. 12, 196.

Bradford, M.M., 1976. A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Anal. Biochem. 72, 248.

Capaldi, R.A., Green, D.E., 1972. Membrane protein and

membrane structure. FEBS Lett. 25, 205.

Gitter, C., 1983. Immunity to ticks. In: Warren, K.S., Boweres, J.Z.

(Eds.), Parasitology ± A Global Prospective. Springer, New

York. p. 220.

Guru, P.U., Dhanda, V., Gupta, N.P., 1976. Cell cultures derived

from the developing adults of three species of ticks, by a

simplified technique. Indian J. Med. Res. 64, 1041±1045.

Jackson, L.A., Opdebeeck, J.P., 1989. The effect of antigen

concentration and vaccine regimen on the immunity induced by

membrane antigens from the midgut of Boophilus microplus.

Immunology 68, 272.

Jackson, L.A., Opdebeeck, J.P., 1990. Humoral immune response

of Hereford cattle vaccinated with midgut antigens of the cattle

tick Boophilus microplus. Parasite Immunol. 12, 141.

Johnston, L.A.Y., Kemp, D.H., Pearson, R.D., 1986. Immunization

of cattle against Boophilus microplus using extracts derived

from adult female ticks: effects of induced immunity on tick

populations. Int. J. Parasitol. 16, 27.

Opdebeeck, J.P., Wong, J.Y.M., Jackson, L.A., Dobson, C., 1988a.

Vaccine to protect Hereford cattle against the cattle ticks,

Boophilus microplus. Immunology 63, 363.

Opdebeeck, J.P., Wong, J.Y.M., Jackson, L.A., Dobson, C., 1988b.

Hereford cattle immunized and protected against Boophilus

microplus with soluble and membrane associated antigens from

the midgut of ticks. Parasite Immunol. 10, 405.

Scott, M.T., Snary, D., 1979. Protective immunization of mice

using cell surface glycoprotein from Trypanosoma cruzi.

Nature 282, 73.

Scott, M.T., Neal, R.A., Woods, N.C., 1985. Immunization of

Marmosets with Trypanosoma cruzi cell surface glycoprotein

(GP 90). Trans. R. Soc. Tropical Medicine Hygiene 79, 751.

Singer, S.J., Nicolson, G.L., 1992. The fluid mosaic model of the

structure of cell membrane. Science 175, 720.

Tizzard, I.R., 1992. Veterinary Immunology: An Introduction, 4th

ed. Saunders, Philadephia, PA.

Wakelin, D., 1984. Immunity to Parasites. Edward Arnald Ltd,

London, 48, p. 71.

Wikel, S.K., 1984. Immunomodulation of host responses to

ectoparasite infestation ± an overview. Vet. Parasitol. 14,

321±339.

Willadsen, P., 1980. Immunity to ticks. Adv. Parasitol. 18, 293±

313.

Wong, J.Y.M., Opdebeeck, J.P., 1989. Protective efficacy of

antigens solubilized from gut membranes of the cattle tick

Boophilus microplus. Int. J. Parasitol. 3, 195.

192 M. Akhtar et al. / Small Ruminant Research 33 (1999) 189±192