Inrernarional Journal,for Printed in Grear Brirorn

Parasitology

Vol. 22, No. 5, pp. 627-630,

1992 0

002&7519/92 $5.00 + 0.00 Pergamon Press Ltd Societyfor Parasitology

1992 Ausrralian

DEMONSTRATION OF THE HUMORAL IMMUNE RESPONSE OF HORSES TO BABESIA CABALL BY WESTERN BLOTTING* REINHARD Institute

of Parasitology,

Hannover

BOSE?

and KERSTIN DAEMEN

School of Veterinary

(Received 20 December

Medicine,

Biinteweg

17, D-3000 Hannover

71, Germany

1991; accepted 6 February 1992)

Abstract-B&z R. and DAEMENK. 1992. Demonstration of the humoral immune response of horses to Babesia caballi by Western blotting. International Journal for Parasitology 22: 627-630. Babesia caballiinfected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. cab& and with sera from non-infected

horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141,112,70,50,48,34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and l/33 sera tested false positive by the complement fixation test (CFT) and immunofluorescence antibody test (IFAT). Thus, further characterization and purification of B. cab&antigens are required to identify target antigens for an improved enzyme immuno assay. Until such an assay is available, Western blotting can provide a specific tool for the diagnosis of B. cuballi infections, particularly in cases of contradicting CFT and IFAT results. INDEX KEY WORDS: Western blotting.

Babesia caballi; antigen characterization;

INTRODUCTION

and Babesia equi are the causative agents of equine babesioses which are endemic in most tropical and subtropical areas of the world; in Europe the distribution extends as far north as northern France and central Russia (Friedhoff, 1982). The detection of carrier animals is of particular significance to prevent the introduction of carrier animals into non-endemic areas (Friedhoff, 1982). Horses to be imported into the U.S.A., Japan, Australia and other countries have to be tested negative for babesioses by the complement fixation test (CFT). It has been reported, however, that the CFT yields a considerable proportion of false negative results, thus not detecting carrier animals (see Tenter & Friedhoff, 1986). Consequently, the combination of CFT and the immunofluorescence antibody test (IFAT) has been recommended for safe diagnosis of carrier animals (Weiland, 1986; Tenter & Friedhoff, 1986). The IFAT, however, results in a large number of false positive results (B&e, unpublished). Additionally, the IFAT is laborious and cannot be standardized. Thus, there is Babesia

caballi

*This paper is dedicated to Professor Dr K. T. Friedhoff the occasion of his 60th birthday (5 June 1992). TTo whom all correspondence should be addressed.

on

SDS-polyacrylamide

gel electrophoresis;

still a need for a highly specific and sensitive serological test ideally amenable to standardization and computer evaluation. As a first step towards this aim we investigated the antibody response of horses against B. caballi antigens by Western blotting. MATERIALS AND METHODS Anfigen preparation. Microaerophilous stationary phase (MASP) cultures (Levy & Ristic, 1980) were initiated from blood of a donor pony infected with the USDA strain (Tenter & Friedhoff, 1986) of B. cab&i (see Miiller & Phipps, unpublished). Established cultures were maintained by continuous cultivation as a 10% erythrocyte suspension in RPMI-1640 containing 1.5 g 1-l NaHCO,, 0.584 g 1-l Lglutamine and 50 I.E. gentamycin, pH 7.2 (RPMI) supplemented with 40% donor horse serum (medium). Cultures were held in 24-well tissue culture plates (No. 76033-05, Flow Laboratories, Meckenheim, Germany) and incubated at 38’C in an atmosphere of 5% CO, in air. Each well was filled with 1 ml ofa 10% erythrocyte suspension with B. cab&-infected erythrocytes; the medium was changed daily. Subcultures were made when the percentage of parasitized erythrocytes (PPE) had reached 4% by transferring 0.2 ml of a well-grown culture to a new well filled with 0.8 ml of fresh donor erythrocytes made up to a 10% suspension in medium. For donor erythrocytes, blood from an uninfected pony was defibrinated by glass beads, washed three

628

R. B&E and K. DAEMEN

FIG. I. Demonstration of Babesia caballi antigens. B. caballiinfected erythrocytes (lanes 1,3,5) or normal equine erythrocytes (lanes 2,4,6) were analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions and Western blotting. The equivalent of approximately 0.2 ~1 of erythrocytes was applied per lane and separated on a linear 0.75 mm 10% gel, stained with colloidal Coomassie Brilliant Blue (lanes I and 2) or electroblotted onto PVDF membrane and reacted with a pool of sera from horses infected with B. caballi (lanes 3 and 4) or not infected with Babesia spp. (lanes 5 and 6). After incubation with a rabbit anti-horse-HRP conjugate immunoreactive bands were visualized using 4chloro-1-naphtol as substrate. Bars on the left indicate position of mol. wt markers; bars in the middle indicate apparent mol. wt of major bands recognized by the immune sera (figures in kDa).

times in RPM1 and stored at 4°C for no longer than 8 days. To obtain sufficient material for blotting studies, one part of a well-grown culture on a 24-well plate was diluted with three parts of fresh donor erythrocytes made up in medium; 4 ml were transferred to each well of a six-well tissue culture plate (No. 76-058-05, Flow Laboratories, Meckenheim, Germany). Medium was changed daily and the erythrocyte suspension harvested after 2 days with a PPE of 4-6%. Erythrocytes were pelleted by centrifugation at 12OOg for 10 min at 20°C and resuspended in RPM1 to a 70% erythrocyte suspension. Infected erythrocytes were purified to about 100% parasitized cells on a two-step Percoll gradient (Bhushan, Miiller & Friedhoff, 1991), washed three times in RPMI, resuspended to a 25% erythrocyte suspension, snapfrozen on dry ice and stored at - 80°C. Erythrocytes from the same donor pony used for the cultures were used as a negative control antigen. For this, defibrinated blood was washed three times with RPM1 and the final sediment of erythrocytes snap-frozen and stored in aliquots at - 80°C.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The system devised by Laemmli (1970) was used for separation of antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antigen samples of 10 ~1 were solubilized in 70 ~1 of sample buffer (50 mt+Tris pH 6.8 containing 4% SDS, 20% glycerol, 100 mMdithiothreitol and 0.1% bromphenolblue), heated to 95°C for 5 min, chilled on ice and centrifuged at 13,OOOg for 10 min at 4’C to remove particulate matter. For analytical gels 5 ~1 of the supernatant of the B. cab& or the control antigen were loaded to a 0.75 mm linear 10% separating gel with a 3% stacking gel cast with 5 mm wide lanes and electrophoresed for 20 min at 70 V and for about a further 60 min at 120 V in an electrophoresis unit (‘Mini Protean 2’, Bio-Rad Laboratories, Richmond, U.S.A.). For preparative gels 60 ~1 of the solubilized antigen was applied to a 70 mm wide lane. Gels were stained with colloidal Coomassie Brilliant Blue (Neuhoff, Arold, Taube & Ehrhardt, 1988) or used for blotting. Western blotting. Proteins were transferred from the gels for 1 h at 100 V onto polyvinyliden-difluorid membranes (PVDF) (Immobilon P, Millipore-Corp., Bedford, U.S.A.) using a commercially available apparatus (Trans-blot module, Bio-Rad Laboratories, Richmond, U.S.A.) filled with Tris-glycine-buffer pH 8.3 with 20% methanol. Membrane strips were cut and transferred to an incubation tray (Hoefer, Atlanta, U.S.A.) placed on a rocking table. To reduce non-specific binding, strips were incubated overnight at room temperature in TBS (50 mM-Tris, 150 mM-NaC1, pH 8.0) containing 1% gelatine. Three 5 min washes with TBS containing 0.05% Tween 20 were followed by a 3 h incubation at room temperature with equine sera diluted l/l00 in TBS with 1% gelatine. Three washes with TBS containing 0.05% Tween 20 were followed by a 1 h incubation with a rabbit anti-horse IgG (H+ L) HRP conjugate (Dianova GmbH, Hamburg, Germany) diluted l/4000 in TBS with I % gelatine. After a further set of washes, blots were developed for 5 min using 4-chloro-1-naphthole as substrate. Equine sera used for Western blotting studies were as follows: (I) sera from ponies 1 week to 1 year after infection by intravenous inoculation of a merozoite stabilate of B. caballi (USDA strain); (2) sera from horses field-infected with B. cab& i.e. horses from Corsica tested negative for B. equi but positive for B. caballi by CFT and IFAT at a serum dilution of 2 l/5 and 2 I/SO, respectively; (3) 33 sera from horses tested false positive by CFT or IFAT, i.e. horses tested positive by either CFT (dilution of 2 l/5) or IFAT (dilution 2 l/80) and tested negative by both tests on at least one follow up bleeding; (4) 100 sera from horses from Germany tested negative for B. equi and B. caballi by CFT and IFAT at a serum dilution of 2 l/5 and 2 I/SO, respectively; (5) sera from ponies experimentally infected with B. equi and positive by CFT (dilution of 2 l/20) and by IFAT at a dilution of 2 l/1280.

RESULTS Babesial antigens could be demonstrated by both SDS-PAGE and Western blotting; major antigen bands recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa (Fig. 1). Significant reactions were not observed with the

B. caballi antigens

629

FIG. 2. Reactivity of equine sera with B. caballi antigen on Western blots. B. cabal/i-infected erythrocytes were solubilized in SDS-buffer, the equivalent of 2.5 ~1 of infected erythrocytes separated on a 0.75 mm 10% gel cast with a 70 mm wide preparative comb and electroblotted onto PVDF membrane. Strips shown were reacted with: (1) sera from three horses experimentally infected with B. caballi(lanes 1-12); blots are shown in groups of four representing sera taken 1,2,3 and 4 weeks after infection for each horse, (2) sera from five horses field-infected with B. caballi (lanes 13-l 7), (3) a pool of high titred B. equi sera (lane IS), (4) sera from three of 100 horses all tested negative for Babesia spp. by CFT and IFAT; the three sera with the strongest reactions are shown (lanes 19-21), (5) a selected serum from 33 horses all tested false positive by either CFT or IFAT, the only serum reacting weakly with B. cabal/i antigen bands is shown (lane 22). Bars on the left indicate position of molecular weight markers (figures in kDa).

negative

control antigen. When tested against B. antigen, two of three ponies experimentally infected, strongly recognized the doublet of B. caballi bands at 48 and 50 kDa 1 week after infection; the third horse being positive on blots 2 weeks after infection (Fig. 2). Strongest reactions were observed 1 month after infection. Thereafter the intensity of the staining gradually decreased with no significant change in the banding pattern. Sera from all experimentally infected horses were still positive more than 1 year after infection, i.e. they recognized the doublet of bands at 48 and 50 kDa and at least one additional major antigen band (data not shown). Similarly, sera from five field-infected horses from Corsica recognized the doublet of bands and at least one further band (Fig. 2, lanes 13-17). None of the field-infected horses recognized the 30 and 34 kDa antigens. The pool of high titred B. equi sera showed only weak cross reactions (Fig. 2, lane 18). Ninetyseven from 100 horses tested negative for Bubesia spp. antibodies by CFT and IFAT showed either no or faint reactions with minor bands not identified as B. caballi antigens. Three horses (Fig. 2, lanes 19-21) revealed weak reactions with the 48 and 50 kDa antigen bands and other bands not identified as B. caballi antigens. One of 33 horses tested false positive caballi

by IFAT or CFT reacted weakly with the 48 and 50 kDa antigen bands (Fig. 2, lane 22). DISCUSSION By Western

blotting

we could

demonstrate

seven

and 48 kDa antigen bands are of diagnostic value since they are recognized by sera from horses experimentally or naturally infected with B. caballi. In contrast, the 34 and 30 kDa antigens are recognized by sera raised against the homologous strain, but not by fieldinfected horses. Thus, these bands probably represent strain-specific antigens. The 50 and 48 kDa antigen bands are most consistently recognized by immune sera. Applying the criterion of these two and any of the 141, 112, and 70 kDa bands being recognized, no false positive results were obtained from 133 equine sera tested, 33 of which tested false positive by CFT or IFAT. Thus, Western blotting can provide an additional diagnostic tool for the diagnosis of B. cabdi infections, particularly in cases of contradicting CFT and IFAT results; the main advantage of Western blotting for diagnostic purposes being its specificity. Three of 100 horses tested negative and one from 33 horses tested false positive by CFT and IFAT reacted with the 48 and 50 kDa antigen bands. As the major

babesial

antigen

bands.

The 141,112,70,50

630

R.

BOSE and

parasitological history of these horses is not known, it is possible that this is due to a present or past infection with B. caballi. However, given the low prevalence of B. caballi infections this is unlikely. Similarly, the reactions are probably also not caused by B. equi infections not detected by CFT and IFAT as even high titred B. equi sera caused only weak cross reactions on Western blots (Fig. 2, lane 18). Thus, these reactions are probably spurious cross reactions. Although weakly recognized by four of 133 sera from horses presumably not infected with Bubesiu spp., antigens contained in the 48 and SOkDa bands are candidate antigens for an improved serological test. Given the large number of babesial antigens, it is likely that the doublet of bands contains not only two antigens but several different antigens. This view is supported by the observation that rabbit sera raised against the 50 kDa band recognize both the 50 and 48 kDa bands, whereas sera raised against the 48 kDa band alone do not cross react with the 50 kDa band (data not shown). Thus, further characterization of the antigens contained in the 48 and 50 kDa bands is required until a single target antigen for an improved serological test can be identified. Acknowledgemenfs-We

wish to thank Prof. Dr K. T. Friedhoff and Dr C. Schelp for review of the manuscript and

K. DAEMEN Mr M. Wolfbagen for technical preparation of photographs.

assistance

with the

REFERENCES BHUSHAN C., MOLLERI. & FRIEDHOFF K. T. 1991. Enrichment of Babesia caballi-infected erythrocytes from microaerophilous stationary-phase cultures using Percoll gradients. Parasitology Research 71: 177-179. FRIEDHOFFK. T. 1982. Die Piroplasmen der EquidenBedeutung fiir den internationalen Pferdeverkehr. Berliner und Miinchener Tieriirztliche Wochenschrift 95: 368-374.

LAEMMLI U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 68&685. LEVYM. G. & RISTICM. 1980. Babesia bovis: continuous in vitro cultivation in microaerophilous stationary phase culture. Science 207: 1218-1220. NEUHOFFV., AROLD N., TAUBED. & EHRHARDT W. 1988. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Elecirophoresis 9: 255-262. TENTERA. M. & FRIEDHOFFK. T. 1986. Serodiagnosis of experimental and natural Babesia equi and B. caballi infections. Veterinary Parasitology 20: 49-61. WEILANDG. 1986. Species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test (CFT), immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA). Veterinary Parasitology 20: 4348.

Demonstration of the humoral immune response of horses to Babesia caballi by western blotting.

Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel ...
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