Revision of the antigenic structure of genus Listeria

FEMS MicrobiologyLetters 67 (1990) 113-120 Publishedby Elsevier

113

FEMSLE03845

Revision of the antigenic structure of genus Listeria J.A. G'arcia, L. Dominguez, V. Briones and M. Blanco, J.F. Fernandez-Oarayzabal and G . Suarez Dpto. Patologla Animal I (Sanidad Animal). Fae. de Veterinaria, Unit,. Complutense. MadruL Spain

Received24 August1989 Revisionreceivedand accepted 18 September 1989 Key words: Listeria sp.; Antigen; Structure

1. SUMMARY O-antigenic structure of genus Listeria was studied, using antisera (obtained from rabbits) against different O-antigens of reference strains of each serovar. The titres of sera were determined by agglutination using antigens of the same reference strains as well. Some differences from the actual scheme were found: serum antifactor-[X gave a lower titrc than expected against antigens 4ab and 6b, while the titre observed against antigen 4h was higher than the expected in this case. Serum antifactor-VlII presented a higher titre than could be expected against antigen 6b. The strains of serovars 4d and 4e used in this experience were impossible to distinguish, and could have been classified in the same serovar. We could not obtain serum antifactor-Xl from serovar 6b after several trials. From these differences we propose some modifications of the current antigenic scheme of genus lAsteria.

Correspondence to: L. Dominguez, Dpto. Patologia Animal l (Sanidad Animal), Fa¢. de Veterinaria, Univ. Complutens¢, 28040-Madrid, Spain.

2. INTRODUCTION Serological studies have been a tool widely used in the classification of many bacterial taxa. Antigenic structure of genus t.Js#eria is one of the currently accepted classification based on serological considerations. Some of the first classifications of this genus was established by Julianell¢, Pens and Schultz [1,2] using a seroecological criterium. These were unreal classifications and they were soon discarded. Paterson I3] proposed a new classification considering O and H antigens of Listeria as Kauffmann-White had done with the genus Salmonella. He established 4 serogroups which have been the basis of the actual antigenic scheme. Further investigations showed differences between strains belonging to the same Paterson's group. New O and H-antigans were responsible of these differences, modifying the original Paterson's scheme. Moreover, isolates of no clinical origins made necessary the establishment of now serovars and new species. The currently accepted antigenic structure is due to Seeliger and Donker-Voet and it is the one that can be found in the 9th edition of Bergey's Manual of Systematic Microbiology (4).

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114 MATERIAL A N D M E T H O D S 3.1. B a c t e r i a l s t r a i n s The strains of L i s t e r i a used were those logical reference: L. m o n o c y t o g e n e s - -

of seroNCTC (National Culture Type Collection) 7973 serovar (sv) 1/2a, N C T C 5105 sv 3a, N C T C 5214 sv 4a, N C I ' C 10528 sv 4ab, N C T C 10527 sv 4b, ATCC (American Type Culture Collection) 19116 sv 4c, N C T C 10888 sv 4d, ATCC 19118 sv 4e, SLCC (Special Listeria Culture Collection) 2482 sv 7. L. i v a n o v i i - - ATCC 19119 sv 5, L M D c-664 sv 5 (Leicester University, Dept. of Microbiology Collection, U.K.). L. i n n o c u a - - N C T C 11288 sv 6a, N C T C 11289 sv 6b, L M D c-645 sv 6b. L. w c l s h i m e r i - - SLCC sv 6a. L. s e e l i g e r i - - CIP (Collection de ]'Institut Pasteur, Paris, France) 100100 sv 1/2b. L. g r a y i - - SLCC. Strains were grown ( 3 7 ° C / 1 8 - 2 4 h) on a nutritive medium composed in g / l : Tryptone (Difco) 10, Lah-Lemco powder (Oxoid) 10, Yeast extract (Oxoid) 3, NaC1 2.5, glucose 5, Na2PO4H 7.12, KPO4H 2 2, agar 15. 3.2. O - a n t i g e n s

O-suspensions were prepared as described by Seeliger and Hbhne [5]. After beating ( 1 0 0 ° C / 1 h), O-suspensions were treated with a 1% trypsin solution (15 m i n / 3 7 ° C [6]. Phenol, at a final concentration of 0.2%, was added to the O-suspensions as preservative.

pressed as the reciprocal of the highest dilution showing agglutination. We considered agglutination as the well in which no button of sedimented antigen was observed. The diluent for sera and antigens was PBS 0.1 M p H 7.2 with 0.005% O-safranin (Sigma) dye to enhance visibility of the sedimented antigen. 3.4. A n t i s e r a

Antisera were obtained by intravenous challenge in New Zealand white rabbits (two per strain) with 4 inoculations (1 × 10 s microorganisms/ml in the first and 1 X 10 s microorganisms/ml in the followings) given with ten days interval. Suspensions for inoculations were prepared from microorganisms grown on the nutritive medium ( 3 7 " C / 1 8 - 2 4 h). We were not able to produce antifactors X and Xi, in these cases a new trial with the same strains but inactivated (heated and trypsinized), in the same conditions, was done. 3.5. F a c t o r s c r a

Factor sera were obtained as previously described by Seeliger and H~hne [5], with the following modifications to reduce cross-reactivity: for obtaining anti-O-Vl, the serum anti-sv 4b was absorbed with antigens 4c and 4a; for anti-O-VII, serum anti-4c with antigens 3a, 4d, 4e and 5; for anti-O-VIII, serum anti-4d with 4b, 6b and 4ab; for anti-O-X, serum anti-5 with 4e. Absorptions were carried out as described by Lersen [8].

3.3. A g g l u t i n a t i o n t e s t s

The quantitative determination of agglutinins was performed using the microtechnique described by Gaulmey [7]. The O-antigens were titrated to determinate their optimal concentration, a critical factor for obtaining reproducible results. Briefly, 100 ~tl of serum (diluted 1/10) was placed in the first well of a row of a V-type Microtiter plate, while the rest were filled with 50 Itl of diluent. Twofold serial dilutions of the sera were made using a micropipette (Titertek). We added subsequently 50 pl of the antigen to each wall, so the final dilutions were twice, going from 1/20 to 1/20480. Plates were incubated overnight at 37°C, and then placed at 4 ° C for at least two hours before reading. The serum titres were ex-

4. RESULTS A N D DISCUSSION The titres found against each antigen with each antiserum before absorptions (Table I) did not allow us to suspect the existence of any difference with the serological classification proposed in the 9th edition of l~rgey's Manual [4]. After absorptions were done the results obtained showed slight differences with that classification (Table 2). The titre of serum anti-factor VIII asainst sv 61) was the lowest considered by us as significant (320), which is in disagreement with the actual antigenic scheme. We observed a drop of the homologous titre, after absorbing with antigen of

of rabbit antisera

1/2b

20480 20480 10240 160 40 160 80 80 160 20480 1280 320 40 160

lila

10240 5120 10240 80 80 80 80 40 160 2560 80 80 80 40

Antigen

3. 10240 1280 2560 160 160 160 640 160 10240 5120 160 80 320 40

10 40 20 20480 640 1280 2560 160 160 5120 5120 2560 640 40

4a

40 40 80 1280 20480 10240 1280 20480 1280 5120 5120 2560 40 40

4b

• O-antigen and antiserum of L welshimeri serovar 6a; •• O-antigen and antiserum of L gray;.

w6. 6b 7 G"

5 6.

4d

3. 4a 4b 4ab 4c

1/2b

lila

Sera

Cross~titrt$

Table 1

20 20 80 1280 5120 20480 2560 10240 1280 640 640 10240 160 20

4.b 20 20 80 5120 320 1280 10240 320 320 640 1280 1280 20 40

4c

80 40 320 1280 20480 10240 1280 20480 2560 5120 80 5120 160 40

4d

6.

160 80 320 2560 320 640 160 80 80 20480 10240 320 20 40

160 40 320 20480 20480 20480 5120 20480 1280 250 320 20480 160 40 40 20 160 1280 5120 10240 2560 10240 5120 640 1280 10240 160 20

4e

160 80 80 10240 1280 1280 5120 320 160 20480 5120 1280 80 80

w6a ...

20480 2560 1280 640 20480 20 40

10240

80 20 320 1280 10240 20480

6b 40 5120 160 160 160 40 160 160 80 1280 20 80 20480 20

G -80 20 160 80 80 320 320 80 40 320 20 80 2560 5120

Table 2

40 2560 160 40 160 80 40 80 160 40 40

40

3a

1/2b

20400 80 40 40 40 40 40 160 80 80

2560 40 80 20 20 20 20 40 40 20 80

Antigens

1/2.

• O-antigen from L. we/shimeri serovar 6a.

IV V+VI VI VII VIII IX X XIII XIV XV

Factor Sera

10 20 640 20 2560 10 2560 80 160 80 40

43 20 80 5120 5120 320 160 20 640 80 20 20

43b

Titres or factor-sera against dirferent Lisleria O-antigens.

20 80 20480 10240 160 160 640 160 40 40 80

4b 20 20 320 20 2560 10 80 160 80 20 20

4c 20 80

40

10240 160 5120 20 160 40 40

20480

4d

20 20 20480 10240 160 5120 20 160 40 40 40

4e

20 160 5120 2560 160 320 20 640 20 20 20

40 80 80 20 20 160 80 10 80 20480

320

63

20 80 1280 20 640 10 1240 160 40 80 10240

w'

40 20 10240 5120 1280 320 30 640 80 20 40

6b 20 80 160 20 20 40 20 80 2560 40 20

SO

80 40 80 80 80 80 20 20 2560 40

G

L welshimeri L monocytogenes L. gray;

L ivanovii L innOCllQ

L monocytogenes L seeligeri L monocylogenes

5 6. 6b 6b

4e

4d

4. 4.b 4b 4<:

1/2b 3

Serovar

1/23

Species

IV

O-anligens

V V V V V V V V V V VI VI

VI VI VI

VI VI

VII VII VII

VII

VII VII

VIII

VIII

vm

VIII VIII

O-Antigen ractors round in the strains used in this experiment based on our resuhs

Table 3

IX

IX

IX

X X

X

X

XII XII

XIII XIV

XV

XVI

II II

O-antigens

• modifications; •• Vazquez et al. (9).

L gray; L Mwrayi

6. 6b

4e

4d

4c

3 4. 4ah 4b

1/2

Serovar

(Ill) (Ill) (Ill) (Ill) (Ill) (III) (III) (III) (III) (III) (III) (III) (III) (III)

IV

(V)

V

(V)

V

(V)

(V) V V V VI VI VI (VI) (VI)

VI VI

(VII) (VII)

VII

VII VII

(VIII) •

VIIJ (VIII) (VIII)

O-antigenic formulation of Listeria serovars, including proposed modifications

Table 4

IX

(IX)'

(IX)

IX

(IX)' (IX) •

X

X

X

XI

XII XII XII

XIII XIV XIV

XV

XVI··

xvn··

119

group 4, when using the not inactivated strain to produce the sera, which did not happen when using the strain previously inactivated. These results suggested that factor VIII could be masked by some cellular structures that would difficult its accessibility to the host's immune system. These cellular structures would be destroyed by the heating and trypsin treatment. However, using the strain LMD c-645 we obtained a good titre against factor VIII, which suggested that these structures could not be present in all the strains of sv 6b. Because of this, we think that factor VIII should also appear at least in brackets in the antigenic structure of sv 6b. According to the current antigenic classification [4[ all the strains of sv 4ab and sv 6b would have factor IX, while this factor would not appear in any strain of sv 4b. In our experience sv 4ab and sv 6b showed a titre unreasonably low (20 in hoth cases) against their serum anti-factor. This fact made us suspect that factor IX could not be present in all the strains of these serovars. On the other hand, factor IX had a titre high enough (640) against sv 4b to make us suspect that this factor could appear in some strains of this serovar. Thus, according to our results, serum anti-factor IX should appear in brackets in the antigenic scheme of serovars 4b, 4ab and 6b. From the actual antigenic scheme serovars 4d and 4e should be differentiated by factor IX, present only in the latter. Nevertheless, as we did not find factor IX in the strains of these serovars used by us, both would have been classified in sv 4d. So, the strains of sv 4e that do not have this factor (as the one used by us) should be included in sv 4d, keeping sv 4e exclusively for those strains having factor IX. Therefore in the antigenic structure of sv 4e factor IX should not appear in brackets. Serum anti-factor X was obtained by Seeliger and Hohne [5] using an antisera against a heated antigen of sv 5 absorbed with strains of group 4 and sv 3a. Using the non-inactivated strain ATCC 19119 (sv 5) to produce this antisera, we observed, after absorption. a drop in the homologous titre that would mean the absence of anti factor X in this serum. However, when this strain was inactivated or when another non-inactivated strain (lMO c-664) was used we were able to obtain

serum anti factor X. This could mean that. in some strains of sv 5, a previous inactivation would be necessary to show perfectly factor X. We were not able to produce antifactor XI, even with two different strains (NCTC 11289 and lMO c-645) and in two different ways (inactIvated and non-inactivated). Both with the non-inactivated and inactivated strain we observed a drop in the titre of the serum antifactor XI against L. innocua sv 6b. that was always very similar to that observed with the antiserum against L. monocytogenes sv 4ab, that do not have this factor. So, we think that factor XI could not be present in all the strains of sv 6b and should. therefore, appear in brackets in its antigenic scheme. From this result serovars 4ab and 6b could be confused, except by the existence of factor VIII found by us in sv 6b. Thus, we think that all the strains of sv 6b should have at least one of these two factors (VIII or XI) in order to be distinguished from strains of sv 4ab. From our results the antigenic structures of the strains used in this experiment are shown in Table 3. Table 4 displays the slight differences proposed to the current antigenic scheme of the genus Listeria.

REFERENCES (1) Julianelle. L.A. and Pons, C.A. (1939) Proc. Soc. Exp. Bioi. 4tl, 362-363. [2] Schultz, E.W .• Terry, M.C., Brice, A.T. and Gebhardt, L.P. (1938) Proc. Soc. Exp. BioI. 38, 605-608. (3) Paterson. J.St. (1940) J. Pathol. Bacteriol. 51, 427-436. [4] Seeliger, H.P.R. and Jones, D. (1986) in Bergey's Manual of Systematic Bacteriology, vol. 2, (Sneath, P.H.A. and Holt. J.G .• Eds.), pp. 1235-1245, Williams and Wilkins, Baltimore, MD. [51 SeeIigcr, H.P.R. and HUhne, K. (1979) in Methods in Microbiology, vol. 13, (Bergan, T. and Norris. J.R .. eds.), pp. 31-49, Academic Press, London. [6] Osebold, J.W., Aalund, A. and Chrisp, C.E. (1965) J. Bacteriol. 48, 25. (7) Gaultney, J.B., Wende, R.D. and Williams, R.P. (1971) Appl. Microbiol. 22. 635-640.

(81 La""n. S.A.. Fedey. J.e. and Jones. W.L. (1974) App!. Microbial. Acad. 27, 1005-1013.

[91 Vazquez-Boland, J.A., Dominguez Rodriguez, L., Fernandez Garayzabal, J.F., Blanco Cancelo. J.L.. G6mez-Lucia, E., Briones Oieste. V. and Suarez Fernandez, G. (1988) J. Appl. Bacterial. 64, 371-378.