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Phospholipase D-neutralization in serodiagnosis of Arcanobacterium haemolyticum and Corynebacterium pseudotuberculosis Infections
Zent.bl. Bakteriol. 288, 463-470 (1998) © Gustav Fischer Verlag
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Phospholipase D-Neutralization in Serodiagnosis of Arcanobacterium haemolyticum and Corynebacterium pseudotuberculosis Infections Boris Skalka \ Ivan Literak 2 ,:-, Pavel Chalupa 3, and Miroslav Votava 1 Department of Microbiology, Masaryk University Medical School and St. Anna Hospital, Brno, Czech Republic 2 Department of Infectious Diseases and Veterinary Epidemiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic 3 Department of Infectious Diseases, Faculty Hospital, Brno, Czech Republic 1
Received May 26, 1998 . Revision received July 14, 1998 . Accepted September 2, 1998
Summary Phospholipase D (PLD) neutralization was used to examine sera of humans (n =40) with a spontaneous infection by Arcanobacterium haemolyticum, sheep and goats (n = 76 and 79 respectively) with a spontaneous infection by Corynebacterium pseudotuberculosis, mice (n = 26) experimentally immunized with PLD from A. haemolyticum (PLD-A) and mice (n = 28) experimentally immunized with PLD from C. pseudotuberculosis (PLD-C). PLD-A and PLD-C were also used as neutralizing antigens. A positive result of neutralization was due to an inhibition of the haemolytic synergism with the equi factor from Rhodococcus equi. The titres of sera neutralizing the homologous PLD were always significantly higher than those neutralizing the heterologous PLD. The proportion of sera that were able to neutralize the homologous PLD in sheep, goats and mice immunized with PLD-A significantly exceeded the proportion of sera that neutralized the heterologous PLD. The antigenic properties of PLD-A and PLD-C were similar but not identical.
* Corresponding author.
464
B. Skalka, 1. Literak, P. Chalupa, and M. Votava
Introduction Phospholipase D (PLD) is an enzyme with toxic effects. It is produced by the pathogenic bacteria, Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum (1,2,5, 15). PLDs of these strains have functional similarities which are demonstrated by haemolytic antagonism with the a and ~ staphylococcal haemolysins (4, 10, 16, 18) and haemolytic synergism with the () staphylococcal haemolysin, CAMP factor of Streptococcus agalactiae and equi factor (EF) of Rhodococcus equi (1, 4, 14). Individual PLDs exhibit not only functional, but also genetic similarities (3, 9). Antigenic properties of PLDs produced in vitro have been used in the serodiagnosis of infections caused by bacteria that produce PLDs in vivo. The antigenic identity of individual strains of C. pseudotuberculosis allows a serodiagnosis of animal infections caused by that species (6, 7, 13). The serodiagnosis of human infections caused by A. haemolyticum has also been described (17). Little is known about antigenic differences between individual PLDs. Cuevas and Songer (3) found different titres using homologous and heterologous antigens in caprine sera following experimental applications of PLD from C. pseudotuberculosis and A. haemolyticum. The objective of our work was to study titres of antisera with homologous and heterologous PLD antigens from C. pseudotuberculosis and A. haemolyticum in spontaneous infections of man, sheep and goats, and after experimental immunization of mice.
Materials and Methods Strains of bacteria used
To obtain strain-specific PLDs, A. haemolyticum CNCTC (Czech National Collection of Type Cultures) Cor 79/80 was used to produce PLD-A and C. pseudotuberculosis CNCTC Cor 17162 to produce PLD-C. Rhodococcus equi CNCTC Cor 91188 was used to produce EF and Staphylococcus aureus CNCTC Mau 126/89 to produce ~ haemolysin (~H). Media (all from Difco Laboratories)
Columbia Broth (CB) was used as the basic liquid medium and for the preparation of a semi-solid medium. Columbia Blood Agar Base with 5 % vlv of sheep erythrocytes washed three times was used as solid medium, Agar Noble (AN) was used for the preparation of a semi-solid medium and Agar Technical with 5 % vlv of sheep erythrocytes washed three times was used as blood agar (BA) for the titration of exosubstances and for neutralization tests. Preparation of bacterial exosubstances
The same method was used in preparing all bacterial exosubstances (PLD-A, PLD-C, EF and ~H). The appropriate strain was cultivated in 0.3 % agar medium made of CB and AN. After 72 h incubation at 37"C, the medium showing bacterial growth was frozen and thawed three times. After the third thawing, the material was centrifuged
Phospholipase D-Neutralization
465
and the supernatant was mixed with acetone at a ratio of 1: 2. To obtain concentrated exosubstance, the precipitate was centrifuged and rehydrated with saline to 1/100th of the original supernatant volume. Concentrated exosubstance activity assay BA in Petri dishes was used for titration of concentrated exosubstances. Circular wells 7 mm in diameter were cut in BA, and the agar blocks were removed. Then, 0.1 ml doses of concentrated exosubstance were placed in the wells in twofold dilutions from 1: 100 to 1: 51200. Haemolytic effects were assessed after 24-48 h incubation at 38°C. The last dilution producing a ring 3 mm wide around the well was considered an activity unit (AU) of the substance tested. ~H gave the direct effect of its haemolysis on the basic BA. EF was titrated on BA with 10 AU of ~H in 1 ml of medium, and the synergistic haemolysis was assessed. PLD-A and PLD-C were titrated on BA with 10 AU of EF in 1 ml of medium, and the effect of haemolytic synergism was assessed. Sera from spontaneous infections Forty human sera from patients with bacteriologically confirmed infection with A. haemolyticum and 76 ovine sera and 79 caprine sera infected with C. pseudotuberculosis, whose positivity had been confirmed earlier (11-13), were used. The manifestations of 40 human patients with A. haemolyticum infection were as follows: tonsillitis in 87.5 %, cervical lymphadenopathy in 81.3 %, exanthema in 75 %, fever above 38°C in 50 %, pharyngitis without tonsillitis in 6.3 %. The exanthema was maculopapular with larger morphae than those seen in scarlatina, occurring mainly on the trunk, neck and extremities. The maximum of exanthema was located on the extremities, namely over the extensor regions of arms and thighs and over large joints (elbows, knees, hips). In one case, erythema occurred in the face. The exanthema caused itching in 5 cases. Two cases showed the maximum of exanthema in the areas typical of scarlet fever (hypogastrium, sides of the trunk, volar areas of the forearm and inner parts of the thighs). Patients were aged from 16 to 23 years. Experimental immunization of mice with PLD To obtain PLD antisera, the immunization of laboratory-bred CD-l mice (Charles River, Prague, Czech Republic) was used. The mice were immunized intra peritoneally with 10 doses of 1000 AU of PLD per dose at four-day intervals. A week after the last dosage, the mice were examined serologically. Thirty mice had been immunized in each group, but only 26 PLD-A immunized and 28 PLD-C immunized mice were included in the tests because in the rest (four and two mice, respectively) we failed to obtain enough serum to allow testing. No signs of disease were observed throughout the experimental immunization in the mice. Neutralization test The sera were inactivated for 30 min at 56°C before testing. The tests were performed using the same types of media as in PLD titration. The wells in the medium were cut in the same way as in exosubstance activity tests. Twofold dilutions of the sera tested were prepared (1:2 to 1: 8192), and an equal amount of corresponding PLD in solution (20 AU in 1 ml) was added to each of them. The
466
B. Skalka, I. Literak, P. Chalupa, and M. Votava
final serum dilution in the mixture was therefore 1: 4 to 1: 16284, with the amount of PLD corresponding to 10 AU in 1 ml. After 60 minutes at room temperature, the mixtures were dispensed into the medium at 0.1 ml per well. Results were assessed after 24-48 h incubation at 3 rc. If the neutralization of the corresponding PLD was positive, the BA around the well did not change. If the PLD neutralization was negative, a ring of total haemolysis around the well was produced due to synergic effect of PLD with EF. The titre was defined as the last serum dilution that produced total neutralization. Each serum was examined three times with both PLD-A and PLD-C. Statistical evaluation
The significance of differences between the sets tested with PLD-A and PLD-C was evaluated by means of the X2 test and the Mann-Whitney U test using the Stat-Plus software package (8).
Results The homologous PLD-A was neutralized by human sera at dilutions 1: 8 to 1: 256, most frequently at dilutions of 1: 16 and 1: 32. It was neutralized by all of the sera. The heterologous PLD-C was neutralized by those sera at dilutions of 1: 4 to 1: 64. In one case, the serum failed to neutralize PLD-C even at dilution of 1 :4. The proportion of the human sera that neutralized homologous PLD-A was not significantly higher than the proportion of the sera neutralizing heterologous PLD-C (X2 test, P > 0.05). The titres of the sera neutralizing homologous PLD-A were significantly higher than the titres of the sera neutralizing heterologous PLD-C (V test, P = 0.01). While all 76 ovine sera neutralized homologous PLD-C (titres 1: 8 to 1 :4096),34 of the sera (45 %) failed to neutralize heterologous PLD-A. The heterologous PLD-A was neutralized by part of the sera at dilutions of 1:4 to 1: 32. The proportion of ovine sera that neutralized homologous PLD-C was significantly higher than the proportion of sera which neutralized heterologous PLD-A (X2 test, P < 0.001). The titres of sera neutralizing homologous PLD-C were significantly higher than the titres of the sera neutralizing heterologous PLD-A (V test, P = 0.01). While all 79 caprine sera neutralized homologous PLD-C (titres 1: 8 to 1 :4096),38 sera (48 %) failed to neutralize heterologous PLD-A. The heterologous PLD-A was neutralized by part of the sera at dilutions of 1 : 4 to 1: 32. The proportion of caprine sera that neutralized homologous PLD-C was significantly higher than that of sera neutralizing heterologous PLD-A (X 2 test, P < 0.001). The titres of the sera neutralizing homologous PLD-C were significantly higher than those of the sera neutralizing heterologous PLD-A (V test, P = 0.01). All 26 serum samples from PLD-A-immunized mice were able to neutralize homologous PLD-A (titres 1 : 46 to 1 : 4096). A total of 4 sera (15 %) failed to neutralize heterologous PLD-C. The other 22 sera neutralized heterologous
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467
PLD-C at dilutions of 1: 4 to 1: 64. The proportion of the sera that neutralized homologous PLD-A was significantly higher than that of the sera that neutralized heterologous PLD-C (X 2 test, P. < 0.05). The titres of the sera neutralizing homologous PLD-A were significantly higher than those of the sera neutralizing heterologous PLD-C (U test, P = 0.01). All 28 sera of the PLD-C immunized mice were able to neutralize PLD-C (titres 1:64 to 1:8192) as well as heterologous PLD-A (titres 1:4 to 1:512). The serum titres at which homologous PLD-C was neutralized were higher than those at which heterologous PLD-A was neutralized (U test, P = 0.01). The results of serological examinations are given in Table 1.
Discussion The sera obtained from spontaneous infection cases reacted with the homologous PLD of the bacterial species which had caused the infection. Nevertheless, their reactivity showed large differences. We cannot offer any definite explanation of such a variation in the reactivity because in neither group we were able to determine the length of infection or the intensity of the antigenic stimulus. All the sera from spontaneous infection cases reacted with the homologous antigen, but only some of them reacted with the heterologous antigen. With the homologous antigen, the sera reacted at higher titres than with the heterologous one. The difference in reactivity of sera from spontaneous infections with homologous and heterologous PLDs indicate that, from the antigenic point of view, PLD-A and PLD-C are similar but not identical. In contrast to spontaneous infection cases, the amount of PLD administered and the length of infection were known in all experimentally infected mice; in other words, the immunized mice represented a relatively homogenous group. Even under those conditions, however, marked individual differences in the serological response were observed. In PLD-A-immunized mice, positive titres in reaction with the homologous antigen comprised seven different dilutions. In PLD-C immunized mice, positive titres with the homologous antigen comprised also seven different dilutions. The range of positive titres in reactions with heterologous antigens, however, was lower. In experimentally immunized mice, the sera also responded to the homologous antigen at higher titres than to the heterologous one. The different reactivity of the sera from experimentally immunized animals with the homologous and heterologous PLDs confirms again that PLD-A and PLD-C are not antigenically identical. Our results confirm and supplement the existing information on antigenic characteristics of PLD-A and PLD-C reported by Cuevas and Songer (3). These authors concluded that the reactivity of antisera obtained by immunization of goats with native PLD-A and PLD-C was either higher or lower, depending on whether homologous or heterologous antigens were neutralized.
Number of sera neutralizing PLD in the titre_
3/52
369/9
32110
<- -
34/0 27/0 10/22 3/7 2/9 0112 0110 0/4 0/4 0/5 0/2 011
0/4 0/3 0/6 0/6 0/6 4/1 4/0 7/0 4/0 3/0 1/0 3/0
0*/1 0112 3/11 13/9 11/5 8/2 4/0 1/0
<4 4 8 16 32 64 128 256 512 1024 2048 4096 8192 Mean geometric titre 3/209
38/0 2110 14116 5/4 117 0/4 0/4 0/3 0/9 0/8 0115 0/9
Goat (n= 79) Spontaneous PLD-AJPLD-C
0/9 0/3 105/1024
2/0 10/2 6/2 7/6 211 0/5
110
Mouse (n = 28) Experimental PLD-AJPLD-C
Corynebacterium pseudotuberculosis Sheep (n = 76) Spontaneous PLD-AlPLD-C
Mouse (n = 26) Experimental PLD-AJPLD-C
Man (n=40) Spontaneous PLD-AJPLD-C
Arcanobacterium haemolyticum
Sera from Contact with PLD Antigen Titer
PLD producer
Table 1. Results of neutralization tests with phospholipases D from Arcanobacterium haemolyticum (PLD-A) and Corynebacterium pseudotuberculosis (PLD-C) and sera tested
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The neutralization of homologous PLDs in the serodiagnosis of A. haemolyticum and C. pseudotuberculosis infections has produced encouraging results and it may be recommended as an easy to perform indirect diagnostic method for A. haemolyticum infections in people and for C. pseudotuberculosis infections in sheep and goats. The method described has been successfully used in the diagnosis of A. haemolyticum infections in humans and of caseous lymphadenitis in sheep and goats in the Czech Republic (11-13). Acknowledgements. The study was funded by grant no. 3714-3 from the IGA of the Ministry of Health of the Czech Republic. We thank Mrs. A. Hornackova for excelent technical assistance.
References 1. Barksdale, L., R. Linder, I. T. Sulea, and M. Pollice: Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis), and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium. J. Clin. MicrobioI. 13 (1981) 335-345 2. Brown, C. C. and H.]. Olander: Caseous lymphadenitis of goats and sheep: A review. Vet. Bull. 57 (1987) 1-12 3. Cuevas, w: A. and]. G. Songer: Arcanobacterium haemolyticum phospholipase D is genetically and functionally similar to Corynebacterium pseudotuberculosis phospholipase D. Infect. Immun. 61 (1993) 4310-4316 4. Fraser, G.: The effect on animal erythrocytes of combinations of diffusible substances produced by bacteria. J. Pathol. Bacteriol. 88 (1964) 43-53 5. Funke, G., A. von Graevenitz,]. E. Claridge III, and K. A. Bernard: Clinical microbiology of coryneform bacteria. Clin. Microbiol. Rev. 10 (1997) 125-159 6. Holstad, G.: Corynebacterium pseudotuberculosis infection in goats. I. Evaluation of two serological tests. Acta Vet. Scand. 27 (1986) 575-583 7.Johnson, E.H., C.E.S. Vidal, and J.S.Rosa: Comparison of a diffusion in gelenzyme-linked immunosorbent assay (DIG-ELISA) and the synergistic haemolysis inhibition assay to record the antibody response of goats to the exotoxin of Corynebacterium pseudotuberculosis. Rev. Microbiol. 19 (1988) 374-378 8. Matouskova, 0.: Stat-Plus (in Czech). Veterinary Research Institute, Brno 1992 9. McNamara, P.]., w: A. Cuevas, and G.]. Songer: Toxic phospholipases D of Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium heamolyticum: cloning and sequence homology. Gene 156 (1995) 113-118 10. Munch-Petersen, E.: A corynebacterial agent which protects ruminant erythrocytes against staphylococcal ~ toxin. Aust. J. Exp. BioI. 32 (1954) 361-368 11. Skalka, B. and P. Chalupa: Serodiagnosis of infections caused by Arcanobacterium haemolyticum (in Czech, English sum.). Klinicka mikrobiologie a infekcn! lekarstV! 4 (1998) 84-87 12. Skalka, B. and 1. Literitk: Serodiagnosis of caseous lymphadenitis (pseudotuberculosis) of sheep (in Czech, English sum.). Vet. Med. - Czech 39 (1994) 533-539 13. Skalka, B., I. Literak, I. Michalik, and M. Skiivanek: Corynebacterium pseudotuberculosis infection in goats in the Czech Republic. J. Vet. Med. B 45 (1998) 3135
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14. Skalka, B., J. Smola, and J. Pillich: Diagnostical availability of the hemolytic ally active exosubstances of Corynebacterium pseudotuberculosis for isolation and identification of Streptococcus agalactiae, and its comparison with the beta toxin of Staphylococcus aureus. Zbl. Vet. Med. B 26 (1979) 679-689 15. Soucek, A., A. Souckova, M. Mara, and F. Patocka: Observations on the biological properties of atypical haemolytic corynebacteria isolated from man as compared with Cor. haemolyticum, Cor. pyogenes bovis and Cor. ovis. II. In vitro investigations. J. Hyg. Epidemiol. Microbiol. Immunol. 6 (1962) 13-23 16. Souckova, A. and A. Soucek: Inhibition of the hemolytic action of alpha and beta lysins of Staphylococcus pyogenes by Corynebacterium haemolyticum, C. ovis and C. ulcerans. Toxicon 10 (1972) 501-509 17. Wickremsinghe, R. S. B.: Corynebacterium haemolyticum infections in Sri Lanka. J. Hyg. (Camb.) 87 (1981) 271-276 18. Zahorova, L. and V. Kubelka: A reverse CAMP diagnostic test with Corynebacterium pyogenes varietas hominis. Folia Microbiol. 5 (1960) 57-59 Corresponding author: Doc. MVDr. Ivan Literak, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-61242 Brno, Czech Republic, Tel.: ++420-5-41562307, Fax: ++420-5-748841, E-mail: literaki @vfu.cz
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Phospholipase D-Neutralization in Serodiagnosis of Arcanobacterium haemolyticum and Corynebacterium pseudotuberculosis Infections Boris Skalka \ Ivan Literak 2 ,:-, Pavel Chalupa 3, and Miroslav Votava 1 Department of Microbiology, Masaryk University Medical School and St. Anna Hospital, Brno, Czech Republic 2 Department of Infectious Diseases and Veterinary Epidemiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic 3 Department of Infectious Diseases, Faculty Hospital, Brno, Czech Republic 1
Received May 26, 1998 . Revision received July 14, 1998 . Accepted September 2, 1998
Summary Phospholipase D (PLD) neutralization was used to examine sera of humans (n =40) with a spontaneous infection by Arcanobacterium haemolyticum, sheep and goats (n = 76 and 79 respectively) with a spontaneous infection by Corynebacterium pseudotuberculosis, mice (n = 26) experimentally immunized with PLD from A. haemolyticum (PLD-A) and mice (n = 28) experimentally immunized with PLD from C. pseudotuberculosis (PLD-C). PLD-A and PLD-C were also used as neutralizing antigens. A positive result of neutralization was due to an inhibition of the haemolytic synergism with the equi factor from Rhodococcus equi. The titres of sera neutralizing the homologous PLD were always significantly higher than those neutralizing the heterologous PLD. The proportion of sera that were able to neutralize the homologous PLD in sheep, goats and mice immunized with PLD-A significantly exceeded the proportion of sera that neutralized the heterologous PLD. The antigenic properties of PLD-A and PLD-C were similar but not identical.
* Corresponding author.
464
B. Skalka, 1. Literak, P. Chalupa, and M. Votava
Introduction Phospholipase D (PLD) is an enzyme with toxic effects. It is produced by the pathogenic bacteria, Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium haemolyticum (1,2,5, 15). PLDs of these strains have functional similarities which are demonstrated by haemolytic antagonism with the a and ~ staphylococcal haemolysins (4, 10, 16, 18) and haemolytic synergism with the () staphylococcal haemolysin, CAMP factor of Streptococcus agalactiae and equi factor (EF) of Rhodococcus equi (1, 4, 14). Individual PLDs exhibit not only functional, but also genetic similarities (3, 9). Antigenic properties of PLDs produced in vitro have been used in the serodiagnosis of infections caused by bacteria that produce PLDs in vivo. The antigenic identity of individual strains of C. pseudotuberculosis allows a serodiagnosis of animal infections caused by that species (6, 7, 13). The serodiagnosis of human infections caused by A. haemolyticum has also been described (17). Little is known about antigenic differences between individual PLDs. Cuevas and Songer (3) found different titres using homologous and heterologous antigens in caprine sera following experimental applications of PLD from C. pseudotuberculosis and A. haemolyticum. The objective of our work was to study titres of antisera with homologous and heterologous PLD antigens from C. pseudotuberculosis and A. haemolyticum in spontaneous infections of man, sheep and goats, and after experimental immunization of mice.
Materials and Methods Strains of bacteria used
To obtain strain-specific PLDs, A. haemolyticum CNCTC (Czech National Collection of Type Cultures) Cor 79/80 was used to produce PLD-A and C. pseudotuberculosis CNCTC Cor 17162 to produce PLD-C. Rhodococcus equi CNCTC Cor 91188 was used to produce EF and Staphylococcus aureus CNCTC Mau 126/89 to produce ~ haemolysin (~H). Media (all from Difco Laboratories)
Columbia Broth (CB) was used as the basic liquid medium and for the preparation of a semi-solid medium. Columbia Blood Agar Base with 5 % vlv of sheep erythrocytes washed three times was used as solid medium, Agar Noble (AN) was used for the preparation of a semi-solid medium and Agar Technical with 5 % vlv of sheep erythrocytes washed three times was used as blood agar (BA) for the titration of exosubstances and for neutralization tests. Preparation of bacterial exosubstances
The same method was used in preparing all bacterial exosubstances (PLD-A, PLD-C, EF and ~H). The appropriate strain was cultivated in 0.3 % agar medium made of CB and AN. After 72 h incubation at 37"C, the medium showing bacterial growth was frozen and thawed three times. After the third thawing, the material was centrifuged
Phospholipase D-Neutralization
465
and the supernatant was mixed with acetone at a ratio of 1: 2. To obtain concentrated exosubstance, the precipitate was centrifuged and rehydrated with saline to 1/100th of the original supernatant volume. Concentrated exosubstance activity assay BA in Petri dishes was used for titration of concentrated exosubstances. Circular wells 7 mm in diameter were cut in BA, and the agar blocks were removed. Then, 0.1 ml doses of concentrated exosubstance were placed in the wells in twofold dilutions from 1: 100 to 1: 51200. Haemolytic effects were assessed after 24-48 h incubation at 38°C. The last dilution producing a ring 3 mm wide around the well was considered an activity unit (AU) of the substance tested. ~H gave the direct effect of its haemolysis on the basic BA. EF was titrated on BA with 10 AU of ~H in 1 ml of medium, and the synergistic haemolysis was assessed. PLD-A and PLD-C were titrated on BA with 10 AU of EF in 1 ml of medium, and the effect of haemolytic synergism was assessed. Sera from spontaneous infections Forty human sera from patients with bacteriologically confirmed infection with A. haemolyticum and 76 ovine sera and 79 caprine sera infected with C. pseudotuberculosis, whose positivity had been confirmed earlier (11-13), were used. The manifestations of 40 human patients with A. haemolyticum infection were as follows: tonsillitis in 87.5 %, cervical lymphadenopathy in 81.3 %, exanthema in 75 %, fever above 38°C in 50 %, pharyngitis without tonsillitis in 6.3 %. The exanthema was maculopapular with larger morphae than those seen in scarlatina, occurring mainly on the trunk, neck and extremities. The maximum of exanthema was located on the extremities, namely over the extensor regions of arms and thighs and over large joints (elbows, knees, hips). In one case, erythema occurred in the face. The exanthema caused itching in 5 cases. Two cases showed the maximum of exanthema in the areas typical of scarlet fever (hypogastrium, sides of the trunk, volar areas of the forearm and inner parts of the thighs). Patients were aged from 16 to 23 years. Experimental immunization of mice with PLD To obtain PLD antisera, the immunization of laboratory-bred CD-l mice (Charles River, Prague, Czech Republic) was used. The mice were immunized intra peritoneally with 10 doses of 1000 AU of PLD per dose at four-day intervals. A week after the last dosage, the mice were examined serologically. Thirty mice had been immunized in each group, but only 26 PLD-A immunized and 28 PLD-C immunized mice were included in the tests because in the rest (four and two mice, respectively) we failed to obtain enough serum to allow testing. No signs of disease were observed throughout the experimental immunization in the mice. Neutralization test The sera were inactivated for 30 min at 56°C before testing. The tests were performed using the same types of media as in PLD titration. The wells in the medium were cut in the same way as in exosubstance activity tests. Twofold dilutions of the sera tested were prepared (1:2 to 1: 8192), and an equal amount of corresponding PLD in solution (20 AU in 1 ml) was added to each of them. The
466
B. Skalka, I. Literak, P. Chalupa, and M. Votava
final serum dilution in the mixture was therefore 1: 4 to 1: 16284, with the amount of PLD corresponding to 10 AU in 1 ml. After 60 minutes at room temperature, the mixtures were dispensed into the medium at 0.1 ml per well. Results were assessed after 24-48 h incubation at 3 rc. If the neutralization of the corresponding PLD was positive, the BA around the well did not change. If the PLD neutralization was negative, a ring of total haemolysis around the well was produced due to synergic effect of PLD with EF. The titre was defined as the last serum dilution that produced total neutralization. Each serum was examined three times with both PLD-A and PLD-C. Statistical evaluation
The significance of differences between the sets tested with PLD-A and PLD-C was evaluated by means of the X2 test and the Mann-Whitney U test using the Stat-Plus software package (8).
Results The homologous PLD-A was neutralized by human sera at dilutions 1: 8 to 1: 256, most frequently at dilutions of 1: 16 and 1: 32. It was neutralized by all of the sera. The heterologous PLD-C was neutralized by those sera at dilutions of 1: 4 to 1: 64. In one case, the serum failed to neutralize PLD-C even at dilution of 1 :4. The proportion of the human sera that neutralized homologous PLD-A was not significantly higher than the proportion of the sera neutralizing heterologous PLD-C (X2 test, P > 0.05). The titres of the sera neutralizing homologous PLD-A were significantly higher than the titres of the sera neutralizing heterologous PLD-C (V test, P = 0.01). While all 76 ovine sera neutralized homologous PLD-C (titres 1: 8 to 1 :4096),34 of the sera (45 %) failed to neutralize heterologous PLD-A. The heterologous PLD-A was neutralized by part of the sera at dilutions of 1:4 to 1: 32. The proportion of ovine sera that neutralized homologous PLD-C was significantly higher than the proportion of sera which neutralized heterologous PLD-A (X2 test, P < 0.001). The titres of sera neutralizing homologous PLD-C were significantly higher than the titres of the sera neutralizing heterologous PLD-A (V test, P = 0.01). While all 79 caprine sera neutralized homologous PLD-C (titres 1: 8 to 1 :4096),38 sera (48 %) failed to neutralize heterologous PLD-A. The heterologous PLD-A was neutralized by part of the sera at dilutions of 1 : 4 to 1: 32. The proportion of caprine sera that neutralized homologous PLD-C was significantly higher than that of sera neutralizing heterologous PLD-A (X 2 test, P < 0.001). The titres of the sera neutralizing homologous PLD-C were significantly higher than those of the sera neutralizing heterologous PLD-A (V test, P = 0.01). All 26 serum samples from PLD-A-immunized mice were able to neutralize homologous PLD-A (titres 1 : 46 to 1 : 4096). A total of 4 sera (15 %) failed to neutralize heterologous PLD-C. The other 22 sera neutralized heterologous
Phospholipase D-Neutralization
467
PLD-C at dilutions of 1: 4 to 1: 64. The proportion of the sera that neutralized homologous PLD-A was significantly higher than that of the sera that neutralized heterologous PLD-C (X 2 test, P. < 0.05). The titres of the sera neutralizing homologous PLD-A were significantly higher than those of the sera neutralizing heterologous PLD-C (U test, P = 0.01). All 28 sera of the PLD-C immunized mice were able to neutralize PLD-C (titres 1:64 to 1:8192) as well as heterologous PLD-A (titres 1:4 to 1:512). The serum titres at which homologous PLD-C was neutralized were higher than those at which heterologous PLD-A was neutralized (U test, P = 0.01). The results of serological examinations are given in Table 1.
Discussion The sera obtained from spontaneous infection cases reacted with the homologous PLD of the bacterial species which had caused the infection. Nevertheless, their reactivity showed large differences. We cannot offer any definite explanation of such a variation in the reactivity because in neither group we were able to determine the length of infection or the intensity of the antigenic stimulus. All the sera from spontaneous infection cases reacted with the homologous antigen, but only some of them reacted with the heterologous antigen. With the homologous antigen, the sera reacted at higher titres than with the heterologous one. The difference in reactivity of sera from spontaneous infections with homologous and heterologous PLDs indicate that, from the antigenic point of view, PLD-A and PLD-C are similar but not identical. In contrast to spontaneous infection cases, the amount of PLD administered and the length of infection were known in all experimentally infected mice; in other words, the immunized mice represented a relatively homogenous group. Even under those conditions, however, marked individual differences in the serological response were observed. In PLD-A-immunized mice, positive titres in reaction with the homologous antigen comprised seven different dilutions. In PLD-C immunized mice, positive titres with the homologous antigen comprised also seven different dilutions. The range of positive titres in reactions with heterologous antigens, however, was lower. In experimentally immunized mice, the sera also responded to the homologous antigen at higher titres than to the heterologous one. The different reactivity of the sera from experimentally immunized animals with the homologous and heterologous PLDs confirms again that PLD-A and PLD-C are not antigenically identical. Our results confirm and supplement the existing information on antigenic characteristics of PLD-A and PLD-C reported by Cuevas and Songer (3). These authors concluded that the reactivity of antisera obtained by immunization of goats with native PLD-A and PLD-C was either higher or lower, depending on whether homologous or heterologous antigens were neutralized.
Number of sera neutralizing PLD in the titre_
3/52
369/9
32110
<- -
34/0 27/0 10/22 3/7 2/9 0112 0110 0/4 0/4 0/5 0/2 011
0/4 0/3 0/6 0/6 0/6 4/1 4/0 7/0 4/0 3/0 1/0 3/0
0*/1 0112 3/11 13/9 11/5 8/2 4/0 1/0
<4 4 8 16 32 64 128 256 512 1024 2048 4096 8192 Mean geometric titre 3/209
38/0 2110 14116 5/4 117 0/4 0/4 0/3 0/9 0/8 0115 0/9
Goat (n= 79) Spontaneous PLD-AJPLD-C
0/9 0/3 105/1024
2/0 10/2 6/2 7/6 211 0/5
110
Mouse (n = 28) Experimental PLD-AJPLD-C
Corynebacterium pseudotuberculosis Sheep (n = 76) Spontaneous PLD-AlPLD-C
Mouse (n = 26) Experimental PLD-AJPLD-C
Man (n=40) Spontaneous PLD-AJPLD-C
Arcanobacterium haemolyticum
Sera from Contact with PLD Antigen Titer
PLD producer
Table 1. Results of neutralization tests with phospholipases D from Arcanobacterium haemolyticum (PLD-A) and Corynebacterium pseudotuberculosis (PLD-C) and sera tested
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Phospholipase D-Neutralization
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The neutralization of homologous PLDs in the serodiagnosis of A. haemolyticum and C. pseudotuberculosis infections has produced encouraging results and it may be recommended as an easy to perform indirect diagnostic method for A. haemolyticum infections in people and for C. pseudotuberculosis infections in sheep and goats. The method described has been successfully used in the diagnosis of A. haemolyticum infections in humans and of caseous lymphadenitis in sheep and goats in the Czech Republic (11-13). Acknowledgements. The study was funded by grant no. 3714-3 from the IGA of the Ministry of Health of the Czech Republic. We thank Mrs. A. Hornackova for excelent technical assistance.
References 1. Barksdale, L., R. Linder, I. T. Sulea, and M. Pollice: Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis), and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium. J. Clin. MicrobioI. 13 (1981) 335-345 2. Brown, C. C. and H.]. Olander: Caseous lymphadenitis of goats and sheep: A review. Vet. Bull. 57 (1987) 1-12 3. Cuevas, w: A. and]. G. Songer: Arcanobacterium haemolyticum phospholipase D is genetically and functionally similar to Corynebacterium pseudotuberculosis phospholipase D. Infect. Immun. 61 (1993) 4310-4316 4. Fraser, G.: The effect on animal erythrocytes of combinations of diffusible substances produced by bacteria. J. Pathol. Bacteriol. 88 (1964) 43-53 5. Funke, G., A. von Graevenitz,]. E. Claridge III, and K. A. Bernard: Clinical microbiology of coryneform bacteria. Clin. Microbiol. Rev. 10 (1997) 125-159 6. Holstad, G.: Corynebacterium pseudotuberculosis infection in goats. I. Evaluation of two serological tests. Acta Vet. Scand. 27 (1986) 575-583 7.Johnson, E.H., C.E.S. Vidal, and J.S.Rosa: Comparison of a diffusion in gelenzyme-linked immunosorbent assay (DIG-ELISA) and the synergistic haemolysis inhibition assay to record the antibody response of goats to the exotoxin of Corynebacterium pseudotuberculosis. Rev. Microbiol. 19 (1988) 374-378 8. Matouskova, 0.: Stat-Plus (in Czech). Veterinary Research Institute, Brno 1992 9. McNamara, P.]., w: A. Cuevas, and G.]. Songer: Toxic phospholipases D of Corynebacterium pseudotuberculosis, C. ulcerans and Arcanobacterium heamolyticum: cloning and sequence homology. Gene 156 (1995) 113-118 10. Munch-Petersen, E.: A corynebacterial agent which protects ruminant erythrocytes against staphylococcal ~ toxin. Aust. J. Exp. BioI. 32 (1954) 361-368 11. Skalka, B. and P. Chalupa: Serodiagnosis of infections caused by Arcanobacterium haemolyticum (in Czech, English sum.). Klinicka mikrobiologie a infekcn! lekarstV! 4 (1998) 84-87 12. Skalka, B. and 1. Literitk: Serodiagnosis of caseous lymphadenitis (pseudotuberculosis) of sheep (in Czech, English sum.). Vet. Med. - Czech 39 (1994) 533-539 13. Skalka, B., I. Literak, I. Michalik, and M. Skiivanek: Corynebacterium pseudotuberculosis infection in goats in the Czech Republic. J. Vet. Med. B 45 (1998) 3135
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B. Skalka, I. Literak, P. Chalupa, and M. Votava
14. Skalka, B., J. Smola, and J. Pillich: Diagnostical availability of the hemolytic ally active exosubstances of Corynebacterium pseudotuberculosis for isolation and identification of Streptococcus agalactiae, and its comparison with the beta toxin of Staphylococcus aureus. Zbl. Vet. Med. B 26 (1979) 679-689 15. Soucek, A., A. Souckova, M. Mara, and F. Patocka: Observations on the biological properties of atypical haemolytic corynebacteria isolated from man as compared with Cor. haemolyticum, Cor. pyogenes bovis and Cor. ovis. II. In vitro investigations. J. Hyg. Epidemiol. Microbiol. Immunol. 6 (1962) 13-23 16. Souckova, A. and A. Soucek: Inhibition of the hemolytic action of alpha and beta lysins of Staphylococcus pyogenes by Corynebacterium haemolyticum, C. ovis and C. ulcerans. Toxicon 10 (1972) 501-509 17. Wickremsinghe, R. S. B.: Corynebacterium haemolyticum infections in Sri Lanka. J. Hyg. (Camb.) 87 (1981) 271-276 18. Zahorova, L. and V. Kubelka: A reverse CAMP diagnostic test with Corynebacterium pyogenes varietas hominis. Folia Microbiol. 5 (1960) 57-59 Corresponding author: Doc. MVDr. Ivan Literak, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-61242 Brno, Czech Republic, Tel.: ++420-5-41562307, Fax: ++420-5-748841, E-mail: literaki @vfu.cz