Evaluation of different serological tests for detection of antibodies against Serpulina hyodysenteriae in pig sera

Comp. Immun. Microbiol. infect. Dis. Vol. 18, No. 3, pp. 215-221, 1995

Pergamon

0147-9571(95)00002-X

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0147-9571/95 $9.50 + 0.00

EVALUATION OF DIFFERENT SEROLOGICAL TESTS FOR DETECTION OF ANTIBODIES AGAINST SERPULINA HYOD YSENTERIAE IN PIG SERA A. T. DIARRA, M. ACHACHA and K. R. MITTAL* Groupe de Recherche sur les maladies infectieuses du pore (GREMIP), Faeult6 de M&tecine V6t6rinaire, Universit6 de Montr6al, Saint-Hyacintbe, Qu6bec, C.P. 5000, Canada J2S 7C6

(Received for publication 12 January 1995) Abstract--Swine dysentery is a mucohemorrhagic diarrheal disease caused by S. hyodysenteriae. The detection of asymptomatic carriers in herds is possible by serological tests. However, cross-reactions between S. hyodysenteriae and S. innocens pose a major problem in serological diagnosis. Several serological tests were evaluated for detection of antibodies to S. hyodysenteriae such as: indirect hemaggiutination, passive hemolysis, conglutination and mieroagglutination tests. Among the tests used, only the microagglutination test was able to detect antibodies to S. hyodysenteriae. 70 to 95% of the pigs were invariably seropositive in a single dilution of 1: 10 in actively infected herds whereas the number of seropositives did not exceed 10% in presumably non-infected herds. The test was found to be simple, and reliable to be used with confidence for detection of herd infection using boiled cell suspension as an antigen.

Key words: Serpulina hyodysenteriae, antibodies, cross-reactions. R ~ 6 - - - - L a dysenteric porcine est une maladie caracteris6e par une diarrh6e mueoh6morragique caus6e par S. hyodysenteriae. La d6tection des pores asymptomatiques dans un troupeau est possible ~i l'aide des tests s6rologiques, cependant, les r6actions crois6es entre S. hyodysenteriae et S. innocens causent un probl6me majeur dans le diagnostic s6rologique. Dans te pr6sent travail, certains tests s6rologiques ont 6t~ 6values, notamment rh~magglutination indirecte, l'h6molyse passive, la congiutination et la microagglutination. Parmi ces tests, seul la microagglutination permet de d6tecter les anticorps dirig~s contre S. hyodysenteriae dans les s6rums de pores infect~s. 70 ~i 95% des pores sont s6ropositifs dans les troupeaux activement infect6s ~i une dilution unique de 1: 10 des s6rums. Cependant le nombre d'animaux infect~s n'exc&te pas 10% dans les troupeaux presumes non-infect6s. La microaggiutination qui s'est av~r6e simple et efficace, pourrait &re utilis6e pour d6celer les troupeaux infect6s.

Mots-clefs: Serpulina hyodysenteriae, anticorps, r6actions crois6es.

INTRODUCTION

Serpulina hyodysenteriae, an anaerobic spirochete, is the causative agent of swine dysentery. S. hyodysenteriae infection is confined to the large intestine in swine and causes severe enteritis with a mucohemorrhagic diarrhea and consequent damage to the large intestine [1-3]. Another spirochete called S. innocens is considered to be a part of the normal flora of swine intestine [4]. The clinical diagnosis is often difficult to establish because of the presence of asymptomatic carriers within the infected herds [5]. The *Author for correspondence. 215

216

A . T . Diarra et al.

detection of asymptomatic carriers in herds is possible by serological tests; however, the major problem is caused by cross-reactions between S. hyodysenteriae and S. innocens. The detection and control of swine dysentery is important to the swine industry to enable proper management of infected herds [2, 6]. Antibodies against S. hyodysenteriae have been detected by a number of different serological assays, such as passive hemolysis [7], agglutination [8, 9], and ELISA [10-12]. However, none of these tests have been free from problems because of lack of sensitivity and/or specificity. There is a need within the swine industry for a serological assay which is simple, specific and sensitive enough to detect S. hyodysenteriae infection in a herd. In this report, indirect hemagglutination, passive hemolysis, conglutination and microagglutination tests were evaluated for detection of antibodies against S. hyodysenteriae in pig sera.

MATERIALS AND METHODS

Bacteria and culture S. hyodysenteriae reference strains representing serotypes 1 (B78), 2 (B204), 3 (B169), 4 (A-l), 5 (B8044), 6 (B6933), 7 (AcK300/8), 8 (FM 88-90) and 9 (FMV 89-3323), and S. innocens (B256) were used. Bacteria were grown on solid media by using blood agar base no. 2 (Oxoid Ltd, Hampshire, U.K.) containing 5% bovine blood. Plates were incubated anaerobically at 42°C for 72 h in jars (Oxoid) by using Gas-pak plus generator atmosphere (BBL, Becton Dickinson and Co., Cokeysville, MD) [13]. Experimental infection and sera from pigs Four to six-week-old piglets were inoculated twice, at 24-h intervals by intragastric intubation and 4 ml of cell suspensions containing 1.1 x 109 cfu/ml of live bacterial cells of reference strains of serotypes I-7 of S. hyodysenteriae and reference strain of S. innocens. The pigs were observed for clinical symptoms. Fecal samples were cultured for isolation of Serpulina as described by Achacha and Messier [13]. Sera samples were collected at day 0 and successively, each week, for 6 weeks post-inoculation. A total of 256 pig sera from several herds, with or without a history of infection, were collected (herds with clinical symptoms, herds with convalescent animals, antibiotic treated, conventional, and specific pathogen free swine). The samples were stored at -20°C until used. Preparation of rabbit hyperimmune sera Antigens for immunization were prepared from 72-h-old cultures on blood agar medium of reference strains of different serotypes of S. hyodysenteriae. The growth from each plate was harvested in 3 ml of phosphate buffer saline solution (PBSS) pH 7.2 containing 0.3% formalin and kept at room temperature for 24 h. A cell suspension adjusted to an optical density (O.D.) of 1 at 540 nm was used for immunization of two rabbits for each serotypes. Briefly, equal volumes of formalin killed bacterial cell suspension and Freund's incomplete adjuvant (Difco) were emulsified and injected into rabbits using the inoculation schedule of Baum and Joens [14]. Rabbits were bled and serum was collected and stored at -20°C until used. Antiserum against serotype 8 (FM 88-90) was found to be most potent and thus used for standardization of serological tests using antigens from all the known serotypes of S. hyodysenteriae.

Antibodies against Serpulina hyodysenteriae in pig sera

217

Serological tests Indirect hemagglutination (IliA) test. The 72-h-old bacterial growth from blood agar was washed in PBS. The suspension was adjusted to an O.D. o f ! at 540 nm and was divided into three parts. The first part was left at room temperature. The second part was boiled for 1 h, and the third was sonicated at 50 Hz for 1 min. Verification of cell desintegration was obtained by dark-field microscopy. All three preparations were centrifuged at 800 g for 30 min. The clear supernatants were referred to as saline, boiled and sonicated extracts respectively. One part of saline extract was treated with 5 parts of chilled absolute alcohol and kept at 4°C overnight. Resulting white precipitate was dissolved in PBSS to the original volume and was referred to as alcohol precipitated antigen. The Westphal hot phenol-water method, adapted by Baum and Joens [14] was used to extract crude lipopolysaccharide (LPS). All five antigen preparations were used to directly coat the sheep red blood cells (SRBCs) for the IHA test. Fresh SRBCs were washed three times in PBSS and were sensitized with antigens as follows. A 0.2 ml of well-washed packed SRBCs was added to a tube containing 2 ml of an optimally standardized dilution of the antigen and the mixture was incubated at 37°C for 1 h. The sensitized SRBCs were again washed three times in PBSS to remove unbound antigen and were suspended in PBSS to a concentration of 1%. The sensitized cells were used on the same day. The IHA test was standardized using rabbit hyperimmune serum produced against reference strain of serotype 8 and SRBCs sensitized with various antigen preparations of the homologous strain. The test was performed in microtiter plates (Dynatech Laboratories Inc.). The serum was heat-inactivated at 56°C for 30min. The heterophile antibodies were removed by adsorption of serum with unsensitized SRBCs at room temperature for 1 h. Serial 2-fold dilutions of serum starting from 1:10 to 1:10,240 were made in 0.05 ml vol. of PBSS in U-bottom microplates. The same volume of sensitized SRBC suspension was added to each well. The contents of the wells were thoroughly mixed by gently tapping the plates. The plates were incubated at 37°C for 2 h before reading. The IHA titer was expressed as the reciprocal of the highest dilution of serum showing a definite positive pattern (fiat sediment) as compared with the pattern of negative control (smooth dot in the center of well). Controls consisted of unsensitized SRBCs plus positive serum, sensitized SRBCs plus negative serum and sensitized SRBCs plus diluent. The same procedure was used to detect antibodies in pig sera against all the known serotypes. Passive hemolysis (PH) test. The test was carried out as described by Jenkins et al. [7]. The test was standardized using rabbit hyperimmune serum produced against a reference strain of serotype 8 and SRBCs sensitized with various antigen preparations of the homologous strain. Guinea pig complement was added to antigen-antiserum mixture. The test was performed in microtiter plates. Briefly, the rabbit serum was heat-inactivated at 56°C for 30 min and adsorbed with SRBCs to remove heterophile antibodies. Serial 2-fold dilutions of rabbit serum from 1 : 10 to 1 : 10,240 were made in 0.05 ml vol. of PBSS in U-bottom microplates. The same volume of sensitized SRBC suspension was added to each well. Guinea pig complement diluted 1:20 was added in 0.05 ml vol. All the necessary controls were kept. The plates were incubated at 37°C for 1 h and observed for lysis, if any. The same procedure was used to detect antibodies in pig sera against all the known serotypes.

A . T . D i a r r a et al.

218

Conglutination test. The test was carried out as described by Coombs et al. [15]. The test was standardized using rabbit hyperimmune serum produced against a reference strain of serotype 8 and SRBCs sensitized with various antigen preparations of the homologous strain. Horse complement and rabbit immunoconglutinin were each added in 0.05 ml vol. Immunoconglutinin was produced in rabbits using kaolin sensitized with horse complement [15]. The test was performed in microtiter plates. Rabbit serum was heat-inactivated at 56°C for 30 min and adsorbed with SRBCs to remove heterophile antibodies. The serum was 2-fold diluted from 1 : 10 to 1 : 10,240 in 0.05 ml vol. in PBSS. SRBCs sensitized with antigen, optimally diluted horse complement and immunoconglutinin were successively added, mixed well, incubated for 1 h at 37°C and observed for conglutination (strong clumping). All the necessary controls were kept. The same procedure was used to detect antibodies in pig sera. Microagglutination test ( M A T ) . The test was carried out as described by Joens et al. [9] and standardized using bacterial cell suspension of a reference strain of serotype 8 as antigen. Bacteria grown on blood agar plates were harvested by centrifugation at 800g; washed once and resuspended in PBSS containing 0.5% formalin and adjusted to an O.D. of 0.3 at 540 nm. The bacterial suspension was divided into 2 aliquots. One portion was kept at room temperature, and the other portion was boiled in a water bath for 1 h. These two preparations referred to as whole cell and boiled cell suspensions respectively were used in MAT. Sera of pigs were heat-inactivated at 56°C for 30 min. Serial 2-fold dilutions of pig sera, beginning at 1:10 to 1:1280 were prepared in 0.05 ml vol. of PBS. The last column was left without serum as a control. An equal volume of test antigen was added to each well. The plates were covered and incubated overnight at 37°C. Antibody titer was expressed as the reciprocal of the highest dilution of serum showing a definite positive pattern (fiat sediment) as compared with the pattern of negative control (smooth dot in the center of well). Pigs showing antibody titer of 10 or more were considered positive.

RESULTS Various tests were evaluated to detect antibody titers in naturally and experimentally infected pig sera. Although indirect hemagglutination, passive hemolysis, and conglutinaTable 1. Comparison of various serological tests for detection of antibodies against S. hyodysenteriae using serotype 8 as antigen Antibody titers in pigs

Test IHA PH Conglt MAT

Infected* Non-infectedt No. of positive pigs/ no. of pigs tested (antibody titer) 0/10 0/10 0/10 10/10

(-) (-) (-) (20-640)

0/10 0/10 0/10 0/10

(-) (-) (-) (-)

*Pigs infected experimentally with S. hyodysenteriae showed clinical symptoms and were culture positive. tPigs (from specific pathogen free farms) did not show any clinical symptoms and were culture negative for S. hyodysenteriae. IHA, indirect hemagglutination; PH, passive hemolysis; Conglt, conglutination test; MAT, microagglutination test; - , negative (antibody titer of < 10).

219

A n t i b o d i e s a g a i n s t Serpulina hyodysenteriae in pig sera Table 2. Detection of antibodies by microagglutination in sera from pigs infected experimentally with S. innocens and different serotypes of S. hyodysenteriae Antibody titers in sera* of pigs infected experimentally with S.i and S. hyodysenteriae Serotypes of S. hyodysenteriae Serotype (antigen)

S.I

l

2

3

4

5

6

7

2 (WC) (BC) 8 (WC) (BC) 9 (WC) (BC)

20 0 20 0 40 0

20 10 20 10 20 10

40 10 20 10 20 10

80 10 80 10 80 10

20 10 20 10 160 10

80 10 40 10 80 l0

40 10 40 10 160 10

20 10 20 l0 20 10

*Sera were obtained from pigs at 4-6 weeks post-infection. S.I, S. innocens; WC, whole bacterial cell suspension; BC, boiled bacterial cell suspension.

tion tests were able to detect antibodies in rabbit hyperimmune serum (results not shown), they failed to detect antibodies in pig sera using any of the sensitizing antigens (Table 1). However, the microagglutination test was able to detect antibodies in both rabbit and pig sera. Higher antibody titers were detected in pig sera when unheated formalinized whole cell suspension was used as antigen (Table 2). Sera of pigs experimentally infected with reference strains representing serotypes 1-7 were tested for the presence of antibodies against various serotypes using unheated and heat treated cells. Cross-reactions were invariably seen among different serotypes of S. hyodysenteriae as well as between S. hyodysenteriae and S. innocens when unheated bacterial cell suspension was used as antigen, however, cross-reactions between S. hyodysenteriae and S. innocens disappeared completely when boiled cell suspension was used as antigen (Table 2). Thus, MAT using boiled cells as antigen was able to differentiate pigs infected with S. hyodysenteriae from those infected with S. innocens on the herd basis. Antigens prepared from any of the strains belonging to serotypes 2, 8, or 9 were found to be equally satisfactory for detection of antibodies against S. hyodysenteriae in MAT (Table 2). Results shown in Table 3 showed that 70-95% of the pigs were seropositive in a single dilution of 1 in l0 in infected herds whereas the number of seropositives varied from 0 to 10% in non-infected herds. MAT detected antibodies in more than 50% of the pigs from herds with a history of infection, whereas less than 10% of pigs in herds known to be free of swine dysentery were seropositive by MAT. Table 3. Detection of antibodies to S. hyodysenteriae by microagglutination test in sera from pigs with various clinical status using boiled cell suspension of serotype 8 as antigen Clinical status of herds* (no. of herds) Herds with clinical symptoms (4) Herds of convalescent swine (I) Herds of swine treated with antibiotic (2) Herds of conventional swine (12) Specific pathogen free (SPF) herds (5)

No. of positive pigst/ no. of pigs tested (% positive) 39/41 (95%)

8/10 (80%) 14/20 (70%)

8/127 (6%) 0•58 (0%)

*S. hyodysemeriae were isolated from pigs in herds with clinical symptoms, convalescent swine, and with antibiotic-treated swine but not from herds of conventional and SPF pigs. tPigs showing antibodies in serum tested in a single dilution of 1 in 10 were considered positive.

220

A, T. Diarra et al. DISCUSSION

The serological methods generally used for serodiagnosis of swine dysentery have been inadequate for the detection of antibodies to S. hyodysenteriae. Among the tests used in the present studies, only MAT was able to detect the presence of antibodies in pig sera. Although the indirect hemagglutination, passive hemolysis and conglutination tests were able to detect antibodies in rabbit hyperimmune serum, they failed to do so in pig sera. There is no satisfactory explanation of this phenomenon. However, it may be speculated that the antibodies produced in response to an infection may apparently be different from those produced in rabbits in response to hyperimmunization with formalinized killed bacterial cells. Moreover, the route of immunization could also play some role in this phenomenon, as the pigs only get infected by an oral route [5]. A variety of serological tests have been reported to detect antibodies to S. hyodysenteriae in the pig sera. Jenkins et al. [7] reported that passive hemolysis is a simple and highly sensitive test for the detection of antibodies against S. hyodysenteriae in pig sera using strain B-78 as antigen. Hunter and Saunder [16] used a tray agglutination test whereas Lee et al. [17] used an indirect fluorescence antibody test. Egan et al. [18] and Smith et al. [11] employed ELISA using whole cell suspension and sonicated extract as antigens respectively. However, none of these tests have been free from problems because of cross-reactions between S. hyodysenteriae and S. innocens. Joens et al. [10] employed ELISA using LPS of serotypes 1 and 2 as antigen and reported that ELISA was more sensitive than MAT and thus would be helpful for detection of positive individual pigs. However, this test being serotype specific, demands knowledge of the prevalence of different serotypes in each geographical area. Joens et al. [9] reported results of MAT using whole cell suspension as antigen and suggested that a titer of 256 or more may indicate a possible exposure of pigs to S. hyodysenteriae. The results of the present investigation (Table 2) clearly indicate that a whole cell-suspension is not a suitable antigen for MAT because of the presence of strong cross-reactions between S. hyodysenteriae and S. innocens, presumably associated with heat labile common antigens. However, the use of boiled cell suspension in MAT proved useful for detection of antibodies against S. hyodysenteriae. Heating of whole cells may destroy several common epitopes. Recent studies have identified a number of outer membrane proteins of S. hyodysenteriae, some of which are shared with S. innocens [19]. Joens et al. [9] reported that MAT antibody titers of pigs infected with S. hyodysenteriae were significantly higher than those in pigs infected with S. innocens. Kinyon et al. [20] reported that S. hyodysenteriae are virulent in swine whereas S. innocens are avirulent. In view of their reports, it is suggested that pigs infected with S. innocens may not be able to provoke a strong immune response in pigs. A combination of different factors, such as low sensitivity of MAT, a higher immune response in pigs to S. hyodysenteriae as compared to S. innocens and the use of boiled cells possessing the minimum possible number of common epitopes as antigen in MAT, instead of using whole cells, permitted us to differentiate herds infected actively with S. hyodysenteriae from those infected with S. innocens. Based on the present results, it is evident that MAT using boiled cell suspension may be simple and reliable for the diagnosis of swine dysentery on a herd basis.

Antibodies against Serpulina hyodysenteriae in pig sera

221

REFERENCES I. Glock R. D. and Harris D. L. Characterisation of lesions in swine innoculated with T. hyodysenteriae in pure and mixed culture. Vet. Med. Small Anita. Clin. 67, 65-69 (1972). 2. Harris D. L. Current status of research on swine dysentery. J. Am. vet. Med. Ass. 164, 809-812 (1974). 3. Taylor D. J. An agent possibly associated with swine dysentery. Res. Vet. Sci. 12, 177-179 (1970). 4. Kinyon J. M. and Harris D. L. Treponema innocens, a new species of intestinal bacteria, emended description of the type strain of Treponema hyodysenteriae. Int. J. Syst. Bacteriol. 29, 102-109 (1979). 5. Alexander T. J. L. and Taylor D. J. The clinical signs, diagnosis and control of swine dysentery. Vet. Rec. 85, 59-63 (1969). 6. Harris D. L. and Lysons R. J. Swine dysentery. In Diseases of Swine (Edited by Leman A. D., Straw B., Mengeling W. L., D'Allaire S. and Taylor D. J.), 7th edition, pp. 599-616. Iowa State Univ. Press, Ames, Iowa (1992). 7. Jenkins E. M., Sinha P. P., Vance R. T. and Reese G. L. Passive hemolysis test for detection of antibody to Treponema hyodysenteriae. Infect. Immun. 14, l l06-1107 (1976). 8. Joens L. A. Comparison of selective culture and serological agglutination of Treponema hyodysenteriae for diagnosis of swine dysentery. Vet. Rec. 105, 463-465 (1979). 9. Joens L. A., Harris D. L., Kinyon J. M. and Kaebede M. L. Microtitration agglutination for detection of Treponema hyodysenteriae antibody. J. clin. Microbiol. 8, 293-298 0978). 10. Joens L. A., Nord N. A., Kinyon J. M. and Egan I. T. Enzyme-linked immunosorbent assay for detection of antibodies to Treponema hyodysenteriae. J. clin. Microbiol. 15, 249-252 0982). 11. Smith S. C., Barrett L. M., Muir T., Christopher W. L. and Coloe P. J. Application and evaluation of enzyme-linked immunosorbent assay and immunoblotting for detection of antibodies to Treponema hyodysenteriae in swine. Epidem. Infect. 107, 285-296 (1991). 12. Wright J. C., Wilt G. R., Reed R. B. and Powe T. A. Use of enzyme-linked immunosorbent assay for detection of Treponema hyodysenteriae infection in swine. J. clin. MicrobioL 27, 411-416 0989). 13. Achacha M. and Messier S. Comparison of six different media for isolation of Treponema hyodysenteriae. J. din Microbiol. 30, 249-251 (1992). 14. Baum D. H. and Joens L. A. Serotypes of beta- hemolytic Treponema hyodysenteriae. Infect. Immun. 25, 792-796 (1979). 15. Coombs R. R. A., Coombs A. M. and Ingram D. G. The Serology of Conglutination and its Relation to Disease. Blackwell, Oxford (1961). 16. Hunter D. and Saunder C. N. Serum agglutination test for swine dysentery. Vet. Rec. 95, 107 0973). 17. Lee C. H., Oldson L. D. and Rodabaugh D. E. Chronology of development of serum antispirochete antibody in swine experimentally exposed to swine dysentery. Am. J. vet Res. 38, 539-540 (1977). 18. Egan I. T., Harris D. L. and Joens L. A. Comparison of microtitration agglutination test and enzyme-linked immunosorbent assay for detection of herds affected with swine dysentery. Am. J. vet. Res. 4, 1323-1328 (1983). 19. Chatfield S. N., Fernie D. S., Penn C. and Gordon D. Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens. Infect. lmmun. 56, 1070-1075 (1988). 20. Kinyon J. M., Harris D. L. and Glock R. D. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect. Immun. 39, 638-646 (1977).