Detection of Antibodies to Babesia equi in Horses by a Latex Agglutination Test Using Recombinant EMA-1

Abstract

A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.

Babesia equi is a tick-borne hemoprotozoan parasite that causes equine piroplasmosis. The disease is characterized clinically by fever, anemia, and icterus. The disease is endemic in most tropical and subtropical areas of the world (8). Due to the almost worldwide distribution of the various tick vectors, the introduction of carriers into areas or countries where the disease is nonendemic must be prevented (8). Prior to importation to such areas or countries, horses must be shown to be negative for piroplasmosis through serological testing (3, 4). The complement fixation test and the indirect fluorescent antibody test (IFAT) are commonly used for detecting B. equi infection. However, these serological tests are generally restricted by antibody detection limits and cross-reactivity (3, 4, 9). Therefore, there is a great need for the development of a rapid, specific, sensitive, and inexpensive serological test.

An immunodominant surface protein, equi merozoite antigen 1 (EMA-1) of B. equi, is considered an important candidate for the development of an effective diagnostic reagent (5, 6). Previously, researchers expressed EMA-1 in insect cells by recombinant baculovirus and demonstrated that the enzyme-linked immunosorbent assay (ELISA) using highly purified recombinant EMA-1 as an antigen is useful for detecting B. equi infection (10). In the present study, we developed a latex agglutination test (LAT) using recombinant EMA-1 expressed in insect cells for the detection of antibodies to B. equi in horses.

The cloning of the EMA-1 gene, construction of recombinant baculovirus AcEMA-1, expression of EMA-1 in insect cells, and purification of secreted EMA-1 have been described elsewhere (10). Purified recombinant EMA-1 was covalently coupled to latex beads according to the methods described previously (7). Carboxylated latex beads (average diameter, 0.9 μm; Sigma, St. Louis, Mo.) were brought to 1% (vol/vol) and were activated overnight at 4°C in phosphate-buffered saline (PBS) containing 2 mg of 1-ethyl-3(3-dimethylamino-propyl) carbodiimide hydrochloride per ml. The activated latex beads were centrifuged at 12,500 × g for 10 min and were washed once with PBS. The washed latex beads were suspended to 1% (vol/vol) in PBS containing 10 μg of EMA-1 per ml. The mixture was allowed to react for 2 h at room temperature on a shaker (200 rpm). The EMA-1-coupled latex beads were centrifuged at 12,500 × g for 10 min and were then suspended to 1% in PBS containing 1% bovine serum albumin. For the latex agglutination procedure, serum or plasma samples were diluted 1:4 to 1:128 with PBS containing 1% bovine serum albumin. The diluted samples (10 μl) were mixed on a glass slide with an equal volume of the EMA-1-coupled latex beads. The slide was rotated by hand for 2 min, and the agglutination was determined visually on the white paper. The test was considered positive when the latex agglutination was observed at a dilution of 1:4 and above. Isolation of B. equi from field samples was done according to the method described previously (1, 2). IFAT and ELISA were performed as described elsewhere (1, 10).

To evaluate whether LAT with recombinant EMA-1 can be used for the detection of antibodies to B. equi in horses, serum samples from horses experimentally infected with either B. equi or B. caballi and from normal horses were tested by LAT. Table 1 shows that all serum samples from 10 horses experimentally infected with B. equi were positive, whereas serum samples from five normal horses or from five horses experimentally infected with B. caballi were negative. In addition, the LAT results were compared with those of the previously developed IFAT (1) and ELISA (10). The LAT results were similar to those of ELISA and IFAT, except that two samples (sera 11 and 14) showed a false positive in IFAT.

TABLE 1.

Comparison of LAT with IFAT and ELISA for detection of antibodies to B. equi in horses experimentally infected with either B. equi or B. caballi and in normal horses

Seruma	Specificity	Reciprocal of dilution ratio for:
		LATb	IFATc	ELISAd
1	B. equi	64	2,560	10,240
2	B. equi	64	1,280	10,240
3	B. equi	64	2,560	5,120
4	B. equi	32	1,280	1,280
5	B. equi	16	320	640
6	B. equi	16	640	640
7	B. equi	16	160	320
8	B. equi	8	320	160
9	B. equi	4	160	80
10	B. equi	4	80	80
11	B. caballi	<4	≤80	<80
12	B. caballi	<4	<80	<80
13	B. caballi	<4	<80	<80
14	B. caballi	<4	≤80	<80
15	B. caballi	<4	<80	<80
16	Normal	<4	<80	<80
17	Normal	<4	<80	<80
18	Normal	<4	<80	<80
19	Normal	<4	<80	<80
20	Normal	<4	<80	<80

^aSerum samples were collected from horses experimentally infected with B. equi or B. caballi (1 to 24 months postinfection) and from normal horses. 

^bLAT was considered positive when agglutination was observed at dilutions of 1:4 and above. 

^cIFAT was considered positive when fluorescence was observed at dilutions of 1:80 and above. 

^dELISA was considered positive when an optical density at 415 nm equal to or greater than 0.1 was observed at dilutions of 1:80 and above. 

Blood samples collected from 40 field horses in central Mongolia (the Ulan Bator region) were investigated by LAT and in vitro culture. As shown in Table 2, 36 (90%) and 12 (30%) samples were identified positively by LAT and in vitro culture, respectively. All 12 (30%) in vitro culture-positive samples were LAT positive. This result indicates that all carrier horses had detectable LAT antibodies. On the other hand, 24 (60%) in vitro culture-negative samples were identified positively by LAT, indicating that some of the LAT-positive horses might have recovered from a previous B. equi infection.

TABLE 2.

Comparison of LAT with in vitro culture method for detection of B. equi infection in field horses

Result given by LAT	No. (%) of results given by culture		Total no. (%) of horses
	Culture positive	Culture negative
Positive	12 (30)	24 (60)	36 (90)
Negative	0 (0)	4 (10)	4 (10)
Total of positive and negative results	12 (30)	28 (70)	40 (100)

The results with five independently produced lots of LAT antigen exhibited nearly perfect reproducibility and agreement in lot-to-lot testing (Table 3). In addition, the LAT antigen kept at 4°C was stable for at least 1 year (data not shown).

TABLE 3.

Reproducibility of LAT antigens among different lots

Serum	Specificity	Reciprocal of dilution ratio given by LAT for:
		Lot 1	Lot 2	Lot 3	Lot 4	Lot 5
1	B. equi	64	64	64	64	64
5	B. equi	16	16	32	32	16
8	B. equi	8	8	16	8	8
11	B. caballi	<4	<4	<4	<4	<4
16	Normal	<4	<4	<4	<4	<4

The results of the present study indicate that highly purified recombinant EMA-1 could be used as an antigen for LAT to provide a simple, rapid, sensitive, specific, and inexpensive alternative to IFAT or ELISA for the detection of antibodies to B. equi in horses. LAT might be very useful in situations in which the time, equipment, and technology required for IFAT or ELISA are not available or are inappropriate.