Intestinal Trichomonads - Journal of Clinical Microbiology - American ...

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Dec 12, 1986 - carried out in tissue culture(RK-13 or McCoy cells), as reported previously (11). Supernatants from 3- to 4-day old cocultures with RK-13 cells ...

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1987, p. 609-614 0095-1137/87/040609-06$02.00/0 Copyright C 1987, American Society for Microbiology

Vol. 25, No. 4

Detection of Hemagglutinins in Cultures of Squirrel Monkey Intestinal Trichomonads FRANK F. PINDAK,î* WILLIAM A. GARDNER, JR.,1 MARGARITA MORA DE PINDAK,2 AND CHRISTIAN R. ABEE2 Departments of Pathology' and Comparative Medicine,2 University of South Alabama College of Medicine, Mobile,

Alabama 36617 Received 29 September 1986/Accepted 12 December 1986

Intestinal trichomonads are very common inhabitants of captive squirrel monkeys. In evaluating the potential pathogenicity of these organisms, we encountered hitherto unknown hemagglutinins in their culture fluids. The cytopathic effect associated with a number of the isolates resembled that caused by vacuolating viruses. We have ruled out conventional viruses as the cause of the cytopathic effect and as the source of the hemagglutinin. The agglutinin has some of the basic characteristics of lectins. Parallel experiments demonstrated agglutination of erythrocytes from squirrel monkeys, humans, dogs, cats, guinea pigs, and horses, with the first two types being the most sensitive. Relatively less agglutination was seen with rat erythrocytes. Agglutination of sheep, rabbit, chicken, and bovine erythrocytes was virtually absent.

Hemagglutinin assay. The agglutination tests were carried out in flat-bottomed, 48-well culture plates (Costar, Cambridge, Mass.). The standard test was performed as follows. Doubling dilutions of culture supernatants (0.8-ml samples)

In an earlier study, we reported a high incidence of intestinal trichomonads in captive squirrel monkeys (11). The organisms were readily detected by direct microscopic examination of fecal suspensions, and, in most instances, dense populations of the protozoa were obtained by cocultivation with tissue cultures. In monolayers of RK-13 cells, the proliferation of some isolates was accompanied by a cytopathic effect (CPE) which was seen as distinct vacuolization resembling that produced by certain vacuolating viruses. While subsequent efforts to demonstrate the presence of a viral agent were unsuccessful, a strongly reactive hemagglutinin was encountered in culture supernatants of many of the isolates. Similarly acting lectins or lectinlike products of other protozoa were previously described (3, 5, 7, 9), but the association of such substances with trichomonads was not recognized. This paper presents data on the common occurrence with intestinal trichomonads of a hemagglutinin and describes some of its basic properties. (Preliminary results of this investigation were presented previously [F. F. Pindak, W. A. Gardner, Jr., M. Mora de Pindak, and C. R. Abee, Abstr. 9th Int. Congr. Infect. Parasit. Dis., abstr. no. 1353, 1986].)

prepared in phosphate-buffered saline (PBS; pH 7.2). Each well received 0.05 ml of erythrocyte suspension containing 109 cells per ml (see below). After being thoroughly mixed, the samples were left undisturbed at room temperature overnight. After resuspension by rotary agitation, the samples were kept on a 45°-slanted platform until the erythrocytes showed definite settling patterns. Control and negative wells settled in a double-convex pattern; positive wells showed a concave (quarter-moon) pattern or an irregular disk (Fig. 1). Test scoring and interpretation. To maintain a degree of uniformity in tests in which numerous isolates were used for agglutination of erythrocytes from various species, the results (Table 1) were measured in units of hemagglutinin in undiluted samples; 1 U was defined as the amount present in the highest dilution of a culture supernatant that showed definite agglutination of erythrocytes. (Thus, supernatant which produced agglutination only when undiluted contained 1 U of hemagglutinin; endpoints of agglutination in 1:2 to 1:16 dilutions indicated 2 to 16 U of hemagglutinin, respectively, in the original supernatant.) Erythrocytes tested. Heparinized whole human type-O blood was obtained from volunteers; squirrel monkey blood and dog blood were collected from healthy animals at our facility; blood from all other species was purchased from Colorado Serum Co., Denver, Colo. Ail erythrocytes were washed three times in 10 volumes of PBS (pH 7.2). The final concentration of 109 cells per ml was determined by actual cell count in a hemacytometer. were

MATERIALS AND METHODS Cultivation of trichomonads for production of hemagglutinin. All strains of intestinal trichomonads studied were isolated in our laboratory from rectal swabs of squirrel monkeys (Saimiri sciureus and Saimiri boliviensis) maintained at the Primate Research Laboratory of the University of South Alabama. The animals were housed and maintained as described elsewhere (2). A number of arbitrarily chosen samples were received from primate colonies maintained at two other locations. Initial recovery of the organisms was carried out in tissue culture (RK-13 or McCoy cells), as reported previously (11). Supernatants from 3- to 4-day old cocultures with RK-13 cells were screened for hemagglutinin. Each supernatant was tested simultaneously against erythrocytes from a variety of species (Table 1). *

RESULTS Monolayers of RK-13 cells inoculated with several isolates of squirrel monkey intestinal trichomonads showed pronounced vacuolization (Fig. 2). This kind of CPE suggested the presence of a vacuolating viral agent. Since the original isolations of the protozoa were made in tissue cultures inoculated with fresh stool samples, occasional recovery of a virus would not seem extraordinary. (Indeed, in four in-

Corresponding author. 609

610

PINDAK ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Units of hemagglutinin in supernatants of 24 isolates, measured by agglutination of erythrocytes of various species Amt (U) of hemagglutinin in isolates from: Isolate no. Squirrel Human Dog Cat Guinea pig Horse Rat Sheep Rabbit monkey 25 4 4 4 0 4 0 2 0 1 48 8 4 2 4 0 4 1 1 1 49 2 4 4 4 0 1 0 2 2 52 81 223

8 8 8

8 8 4

8 8 2

8 8 1

8 8 8

1 8 8

1 1 1

0 1 0

0 0 1

227 230 231

0 8 8

0 8 8

0 8 8

0 8 8

0 8 8

0 8 8

0 8 8

0 0 1

0 1 1

233

234b 236

8 8 8

8 8 8

8 8 8

8 8 4

8 8 8

4 4 8

8 4 8

1 1 1

1 1 2

241b 307 470

8 8 4

8 8 8

8 8 8

8 8 8

8 8 8

8 4 2

4 8 2

0 1 1

0 1 0

688 776b 820

8

8

8

8

8

4

4

1

1

0

1

1

1

0

1

0

0

0

8

8

8

8

8

8

1

1

0

911 1220 1362

8 8 4

4 4 4

4 4 1

4 2 1

8 4 2

8 8 8

4 i 1

1 1 0

0 0 0

1370 1376

4 8 1

8 8 4

8 8 2

8 8 2

2 2 1

1 2 1

1 1 1

0 0 0

0 0 0

4600b a

b

Results with bovine and chicken erythrocytes are not included in the table. Tests with these were largely negative. Isolates with proven CPE in RK-13 cells.

B

A

2

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