Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, OCt. 1976, p. 1106-1108
Copyright © 1976 American Society for Microbiology
Passive Hemolysis Test for Antibody to Treponema hyodysenteriae E. M. JENKINS,* P. P. SINHA, R. T. VANCE, AND G. L. REESE
Department of Microbiology, School of Veterinary Medicine, Tuskegee Institute, Tuskegee, Alabama 36088
Received for publication 5 May 1976
Swine were infected orally with pure cultures of Treponema hyodysenteriae, and the humoral antibody response was measured by a passive hemolysis test (passive hemagglutination test with the use of complement). Antibody to T. hyodysenteriae was detected as early as 1 week and later on at 4 months after exposure. However, peak serum titers were obtained after challenge at 4 weeks postinfection. The usefulness of the test as a potential diagnostic tool for antibody to T. hyodysenteriae is discussed. Recent investigations on the etiology of swine dysentery (SD) has indicated that a spirochete, Treponema hyodysenteriae, is most likely the major cause of the disease (4, 6, 12) and not Vibrio coli as previously postulated (3, 8). Since this important discovery, only a limited number of serological tests for the identification of antibody in swine dysentery have been described. A serum agglutination test was described which was thought to be useful as a diagnostic tool for SD on a herd basis rather than for individual pigs (6). Fluorescent antibody tests have been reported that involve the staining of spirochetes in fecal and gut contents for the diagnosis and confirmation of the disease (1, 11, 13). This investigation describes the usefulness of a passive hemolysis test (passive hemagglutination test with the use of complement) for the detection of antibody in the sera of swine experimentally infected with pure cultures of T. hyodysenteriae. T. hyodysenteriae, strain B-78, kindly supplied by D. L. Harris (College of Veterinary Medicine, Iowa State University, Ames), was used. The organism was propagated according to a method previously described (9). Ten female pigs were exposed to T. hyodysenteriae by mixing pure cultures of the organisms with small portions of the feed ration after the animals had fasted overnight. Four weeks after the initial exposure, the pigs were reexposed in the same manner. Conventional YorkshireHampshire cross-bred pigs approximately 8 weeks old were used. A mucoid to mucohemorrhagic diarrhea characteristically seen in SD was used as the main clinical sign for the establishment of infection. Blood samples for sera were collected before and at weekly intervals after exposure.
The sera were processed, divided into equal parts, and stored at -60°C until tested. The antibody titer of sera to T. hyodysenteriae was determined by a modification of a previously described passive hemagglutination test with the use of complement (10). Briefly, twofold serial dilutions of sera (0.5 ml) were mixed with 1 ml of a 0.25% suspension of T. hyodysenteriae-synthesized sheep erythrocytes (SRBC). Guinea pig complement (Difco) was diluted to give maximum hemolysis (1:25), and 0.5 ml was added to all tubes, resulting in a final test volume of 2 ml. The tubes were vigorously shaken, and the material was incubated at 370C for 90 min with occasional shaking. Intact cells were removed by centrifugation for 10 min at 2,000 x g; optical density of the supernatant fluid was measured in a Beckman model DB spectrophotometer at 540 nm. However, comparable results were obtained with the use of a Spectronic 20. Veronal-buffered saline (VBS), prepared by a method previously described (2) with the addition of 0.08% gelatin as a stabilizer, was used as diluent throughout. A complete hemolysis standard was made by the addition of 1 ml of distilled water to the same test volume of SRBC suspended in VBS, and the 50% hemolysis point was determined. Titers of sera were expressed as the last dilution of serum resulting in 50% lysis of the indicator cells. Correction for spontaneous lysis was made by subtracting the optical density of control SRBC supernatant from the test values. Before assay, sera were routinely absorbed with an equal volume of 10% SRBC and incubated for 10 min at room temperature to remove any heterophil antibody. Sensitization of SRBC with T. hyodysenteriae was carried out at 370C for 90 min. Before
1106
VOL.. 14, 1976
NOTES
use, broth cultures of the organisms were inactivated with formalin to a concentration of 0.66% for 24 h at 4°C. The cells were washed three times with VBS and adjusted to 65% transmission at 625 nm on a Spectronic 20. The cell suspension was added to packed SRBC at a ratio of 2:1. The specificity of the antibody was determined by serum absorption tests. Briefly, sera were mixed with dense suspensions of T. hyodysenteriae and incubated at 40C for 24 h. In a similar manner, sera were absorbed with various isolates of enteropathogenic Escherichia coli (EEC) and Salmonella sp. recovered from field cases of swine enteritis. A reduction in the serum titer by homologous organisms and failure of heterologous antigens to reduce the serological activity indicated specificity. A mucoid to mucohemorrhagic diarrhea indicative of SD occurred within 7 to 10 days after exposure and lasted from 48 to 72 h. The diarrhea was less severe upon challenge, which suggested that some resistance to the organism was developed. T. hyodysenteriae was isolated from fecal material and from colonic scrapings of two pigs sacrificed during the course of the
study. The humoral antibody response of the 10 pigs is shown in Table 1. Antibody to T. hyodysenteriae was detected as early as 1 week postinfection. However, the peak serological response was observed at approximately 6 weeks postinitial infection (PII) (2 weeks after challenge) and tended to level off thereafter. The pigs usually recovered by 7 weeks PH; the animals were held over to determine the humoral antibody level at maturity. When retested 4 months PII as mature gilts, an antibody titer of 1:128 was observed, compared with
INFECTION AND IMMUNITY, OCt. 1976, p. 1106-1108
Copyright © 1976 American Society for Microbiology
Passive Hemolysis Test for Antibody to Treponema hyodysenteriae E. M. JENKINS,* P. P. SINHA, R. T. VANCE, AND G. L. REESE
Department of Microbiology, School of Veterinary Medicine, Tuskegee Institute, Tuskegee, Alabama 36088
Received for publication 5 May 1976
Swine were infected orally with pure cultures of Treponema hyodysenteriae, and the humoral antibody response was measured by a passive hemolysis test (passive hemagglutination test with the use of complement). Antibody to T. hyodysenteriae was detected as early as 1 week and later on at 4 months after exposure. However, peak serum titers were obtained after challenge at 4 weeks postinfection. The usefulness of the test as a potential diagnostic tool for antibody to T. hyodysenteriae is discussed. Recent investigations on the etiology of swine dysentery (SD) has indicated that a spirochete, Treponema hyodysenteriae, is most likely the major cause of the disease (4, 6, 12) and not Vibrio coli as previously postulated (3, 8). Since this important discovery, only a limited number of serological tests for the identification of antibody in swine dysentery have been described. A serum agglutination test was described which was thought to be useful as a diagnostic tool for SD on a herd basis rather than for individual pigs (6). Fluorescent antibody tests have been reported that involve the staining of spirochetes in fecal and gut contents for the diagnosis and confirmation of the disease (1, 11, 13). This investigation describes the usefulness of a passive hemolysis test (passive hemagglutination test with the use of complement) for the detection of antibody in the sera of swine experimentally infected with pure cultures of T. hyodysenteriae. T. hyodysenteriae, strain B-78, kindly supplied by D. L. Harris (College of Veterinary Medicine, Iowa State University, Ames), was used. The organism was propagated according to a method previously described (9). Ten female pigs were exposed to T. hyodysenteriae by mixing pure cultures of the organisms with small portions of the feed ration after the animals had fasted overnight. Four weeks after the initial exposure, the pigs were reexposed in the same manner. Conventional YorkshireHampshire cross-bred pigs approximately 8 weeks old were used. A mucoid to mucohemorrhagic diarrhea characteristically seen in SD was used as the main clinical sign for the establishment of infection. Blood samples for sera were collected before and at weekly intervals after exposure.
The sera were processed, divided into equal parts, and stored at -60°C until tested. The antibody titer of sera to T. hyodysenteriae was determined by a modification of a previously described passive hemagglutination test with the use of complement (10). Briefly, twofold serial dilutions of sera (0.5 ml) were mixed with 1 ml of a 0.25% suspension of T. hyodysenteriae-synthesized sheep erythrocytes (SRBC). Guinea pig complement (Difco) was diluted to give maximum hemolysis (1:25), and 0.5 ml was added to all tubes, resulting in a final test volume of 2 ml. The tubes were vigorously shaken, and the material was incubated at 370C for 90 min with occasional shaking. Intact cells were removed by centrifugation for 10 min at 2,000 x g; optical density of the supernatant fluid was measured in a Beckman model DB spectrophotometer at 540 nm. However, comparable results were obtained with the use of a Spectronic 20. Veronal-buffered saline (VBS), prepared by a method previously described (2) with the addition of 0.08% gelatin as a stabilizer, was used as diluent throughout. A complete hemolysis standard was made by the addition of 1 ml of distilled water to the same test volume of SRBC suspended in VBS, and the 50% hemolysis point was determined. Titers of sera were expressed as the last dilution of serum resulting in 50% lysis of the indicator cells. Correction for spontaneous lysis was made by subtracting the optical density of control SRBC supernatant from the test values. Before assay, sera were routinely absorbed with an equal volume of 10% SRBC and incubated for 10 min at room temperature to remove any heterophil antibody. Sensitization of SRBC with T. hyodysenteriae was carried out at 370C for 90 min. Before
1106
VOL.. 14, 1976
NOTES
use, broth cultures of the organisms were inactivated with formalin to a concentration of 0.66% for 24 h at 4°C. The cells were washed three times with VBS and adjusted to 65% transmission at 625 nm on a Spectronic 20. The cell suspension was added to packed SRBC at a ratio of 2:1. The specificity of the antibody was determined by serum absorption tests. Briefly, sera were mixed with dense suspensions of T. hyodysenteriae and incubated at 40C for 24 h. In a similar manner, sera were absorbed with various isolates of enteropathogenic Escherichia coli (EEC) and Salmonella sp. recovered from field cases of swine enteritis. A reduction in the serum titer by homologous organisms and failure of heterologous antigens to reduce the serological activity indicated specificity. A mucoid to mucohemorrhagic diarrhea indicative of SD occurred within 7 to 10 days after exposure and lasted from 48 to 72 h. The diarrhea was less severe upon challenge, which suggested that some resistance to the organism was developed. T. hyodysenteriae was isolated from fecal material and from colonic scrapings of two pigs sacrificed during the course of the
study. The humoral antibody response of the 10 pigs is shown in Table 1. Antibody to T. hyodysenteriae was detected as early as 1 week postinfection. However, the peak serological response was observed at approximately 6 weeks postinitial infection (PII) (2 weeks after challenge) and tended to level off thereafter. The pigs usually recovered by 7 weeks PH; the animals were held over to determine the humoral antibody level at maturity. When retested 4 months PII as mature gilts, an antibody titer of 1:128 was observed, compared with
