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Mechanized procedures for the Serology of Salmonella
Zbl. Bakt. Hyg., I. Abt. Orig. A 255, 258-264 (1983)
Mechanized Procedures for the Serology of Salmonella Mechanisierte Verfahren fiir die Serologie von Salmonellen P. A. M. GUINEE, W. H. JANSEN, and H. M. E. MAAS Laboratory for Bacteriology, National Institute of Public Health, Bilthoven, The Netherlands
With 1 Figure' Received February 28, 1983
Abstract A mechanized microtechnique originally designed for the serotyping of E. coli was adapted to the specificity control of diagnostic Salmonella agglutinating antisera as well as for the serotyping of Salmonella. Salmonella strains sent for serotyping were inoculated into a tube with broth which was used as H antigen and, after heating also as 0 antigen. The agglutination reactions were carried out in clear plastic trays with U formed wells. The antigens were stained with gentian violet in order to obtain a better contrast. With only 6 monofactor 0 sera, 97% of 100.000 cultures received for serotyping in 1979-1982 could be 0 antigen typed. Eight H antisera were sufficient for the complete serotyping of 27% of these strains whereas 64% was partly typed. The technique has a high degree of versatility.
Zusammenfassung Eine mechanisierte Mikrotechnik, die urspriinglich fiir die Serotypisierung von E. coli entwickelt ist, wurde fiir die Oberwachung der Spezifizitat von diagnostischen Salmonellenagglutinierenden Antiseren und fiir die Serotypisierung von Salmonellen modifiziert. Zur Serotypisierung eingeschickte Salmonellenstamme wurden in ein Rohrchen mit Bouillon geimpft. Nach Inkubation wurde diese Keimsuspension als H-Antigen verwendet und dann - nach Erhitzen - auch als O-Antigen. Die Agglutinationsreaktionen wurden in durchsichtigen Mikrotiterplatten mit U-formigen Vertiefungen ausgefiihrt. Die Antigene wurden mit Gentianaviolett gefarbt, urn einen besseren Kontrast zu erreichen. Mit nur 6 Monofaktor-O-Seren konnten 97% von 100.000 Kulturen, die von 1979-1982 zur Serotypisierung eingegangen sind, O-Antigen typisiert werden . 8 H-Antiseren geniigten zur vollstandigen Serotypisierung von 27% dieser Stamme, wahrend 64% zum Teil typisiert wurden. Das Verfahren ist sehr vielseitig verwendbar.
1. Introduction We have reported previously on attempts to develop an automatic device for serotyping of Salmonella (4). Unfortunately the device did not become operational.
Serology of Salmonella
259
This was mainly due to the fact that bacterial contamination of the reagents in the dispensing needles and tubings could not be sufficiently prevented. On the other hand, the mechanized microtechnique for the 0 antigen typing of E. coli was very successful (3). This technique could also be employed for the specificity control of agglutinating antisera and, in a later stage, for the serotyping of Salmonella. In this paper, we describe the procedures finally adopted for the control of antisera and for serotyping. The results of serotyping 21670 Salmonella strains in 1981 are reported.
2. Materials and Methods 2.1. Buffers, stains and medium Formalinized phosphate-buffered saline (FPBS): 7.2 ml of 35% formalin; NaCl, 2 g; Na2HP04·2H20, 1.26 g; KH 2PO" 0.42 g; distilled water, 1000 ml. Gentian violet stock solution (stock): solution of 1% gentian violet in distilled water. 0.4% gentian violet solution: 4 parts of stock and 6 parts of FPBS. 0.004% gentian violet solution: 4 ml of stock in 1000 ml FPBS. 0.002% gentian violet solution: 2 ml of stock in 1000 ml FPBS. One liter of home-made broth is prepared from fat-free beef, 125 g to which is added peptone (Difco), 2.5g; NaCl, 1.25g; 15% (w/v) solution of Na 2C0 3 ·1·H 2 0, 4ml and NaOH 5 M, 3 ml; pH 7.5. Salmonella strains cultured in home-made broth overnight in a shaking waterbath at 37°C yield an OD 600 of about 1.
2.2. Production of antisera The 0 antisera used were prepared and absorbed with standard methods (2,5) according to the immunization scheme of Schlecht and Westphal (6). 05 antiserum was produced with a formalinized suspension of a non-motile variant of S. paratyphi B (Le Minor, personal communication). The H antisera were prepared with the method described by Bader and Winkler (1).
2.3. Control of antisera The multiple dropper containing 120 syringes as originally described (3) has been replaced by an apparatus commercially built to our specifications. This dropper delivers 0.033 ml instead of 0.037 ml by the original one so that about 55 trays (Conphar 120, Continental Pharma, Zutphen, The Netherlands) can be filled from a filling block. The filling block previously described (3) contained 120 separate reservoirs in 8 (A-H) rows of 15 (1-15) so that 120 different antisera could be dispensed (Fig. 1). The mechanized technique can be employed for the specificity control of antisera in a variety of ways. The extreme possibilities per tray are that one antiserum is tested against 120 different antigens or that 120 antisera are tested against one antigen. An intermediate possibility of titrating 15 antisera against one antigen turned out to be very practical. For the extreme possibility mentioned above, two sets of 120 "standard" antigens were designed comprising all Salmonella 0 and H antigens respectively. The "standard" 0 set comprised all 0 antigens in duplicate, except 054, 55 and 67 which were single, and the 0 antigens of Salmonella 0 group A-G which were in triplicate. The "standard" H set comprised all H antigens and the frequently encountered H antigens in 2- to 4-fold. Each antigen was prepared from a different serovar strain. Unabsorbed antisera were tested in the serum dilutions 1: 50 and 1: 100, absorbed sera in the dilutions 1: 25 and 1: 50. Flagellar antisera were tested against the 0 and H set, 0 antisera only against the 0 set unless the antiserum had been prepared with unheated bacteria, e.g. 05 antiserum prepared with formalinized S. paratyphi B suspension. The optimal density of the antigens was determined by checkerboard titrations
260
P.A.M.Guinee, W.H.]ansen, and H.M.E.Maas
A
B C D
E F G
H
0 0 0 0 0 0 0 0
2
3
4
5
6
7
8
9
10 11 12 13 14 15
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Fig. 1. Filling block and tray containing 120 reservoirs and cups respectively.
testing dilutions of Hand 0 antigens against decimal dilutions of antisera. All antigens were stained by adding an equal volume of 0.004% gentian violet solution (final concentration 0.002%). H antigens with an OD600 of 0.8 before staining gave strong and high titred agglutinations after 4-5 h incubation at 45°C. Negative reactions are indicated by a sharp point, positive reactions by a carpet as described before (3). Positive H reactions tend to develop a point after prolonged incubation. The optimal density for 0 antigens was 0.4 before staining when incubated at 37°C. The reactions can be read after 6-20 h incubation at 37°C. The trays were filled with 50 microliter of one dilution of the antiserum to be tested using a dropping pipette (one drop = 50 microliter, Sterilin M36). The antigens were delivered by means of the multiple dropper. If necessary, the syringes of the dropper were rinsed with diluted acetic acid to remove the stain followed by rinsing with water. When using the afore-mentioned intermediate possibility for testing antisera, 15 different antisera were titrated against the standard antigens. The antisera were diluted manually (1: 10-1: 1280 in FPBS) and the dilutions applied to the reservoirs A-H of row 1, 2, 3 etc. of one or more filling blocks (Fig. 1). Each standard antigen was added to one tray by means of a dropping pipette. 2.4. Choice and control of antisera to be used in mechanized serotyping The choice of antisera to be used was based on several considerations: (i) the frequency distribution of the various serotypes in The Netherlands; (ii) the form of the tray to be used: 8 strains against 15 antisera or 15 strains against a maximum of 8 antisera; and (iii) the dilution and concomitantly the "price" of the antisera. The most frequently found Table 1. Most frequently found Salmonella serotypes in The Netherlands between 1970 and 1980 typhimurium panama enteritidis infantis S.hadar S. oranienburg S. montevideo S. newport S.agona S. bovis morbificans
S. S. S. S.
1,4, (5), 12 : i : 1,2 1,9,12 : I, v : 1,5 1,9,12 :g,m 6, 7 :r : 1,5 6,8 : zlO : e, n, x 6,7 : m, t 6,7 : g, m, s:6,8 : e, h : 1,2 : f, g, S 4, 12 6,8 : 1,5 :r
Serology of Salmonella
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serotypes in The Netherlands are given in Table 1. Serotypes belonging to 0 group E1 (= 03,10) do not rank in Table 1, but total up to about 4%. Therefore, the following 60 antisera and 8 H antisera are used: 04, OS, 07, 08, 09, 010 and i, r, v, Z10, 2, 5, "E complex" and "G complex". "E-complex" consists of e, hand e, n, x, z17 antiserum and "G complex" stands for the combination of g, p and m, t antiserum. All other antisera are so-called monofactor antisera; they react with only one antigenic determinant. In addition to the control procedures already described, these antisera underwent extra controls. 0 antisera were tested in trays against 10 strains recently received for serotyping (= routine strains) possessing the homologous 0 antigen. The dilution to be used for serotyping was half of the lowest titer found with any of the 10 routine strains and reduced to a manageable dilution. For example, a particular batch of monofactor 07 antiserum gave titres with 10 routine strains ranging from 160-1280. The manageable dilution chosen was 1: 50. As will be described in the next paragraph, the H antigens of strains to be serotyped have a density of about 1 for practical reasons. With such antigens the incubation time could be reduced to 2'/2 h. The titers obtained under these conditions are one or two steps lower. The H antisera to be used for mechanized serotyping were therefore tested also against H antigens prepared from 10 routine strains under the conditions described above. These 10 strains had previously been found to react in such an antiserum in the slide agglutination using the growth in the condens water of an agar slant culture as antigen. The dilution giving optimal reactivity with as many routine cultures as possible was chosen, provided that it was negative with all heterologous standard monophasic H antigens. Details of the antisera as they have been used for mechanized serotyping over 4 years, are given in Table 2. Filling blocks were adapted to dispense 6-8 antisera in rows of 15 by interconnecting each row of wells (1-15). Filling blocks were closed with adhesive tape
Table 2. Antisera used for the mechanized serotyping of Salmonella strains antiserum
produced with
absorbed
dilution used
approximate quantity of undiluted antiserum required annually
04 05 07 08 09 010
S. bredeney 1 h 100 DC S. paratyphi B formalinized S. infantis 1 h 100 DC S. virginia 1 h 100 DC S. goettingen 1 h 100 DC S. muenster 1 h 100 DC
+ + +
150 x 50X 50x 100X 75 x 75 x
7 ml 20ml 20ml 10 ml 13 ml 13 ml
Hi Hr H(v) H z10 H(2) H(5) "E-complex" "G-complex"
S. bonariensis' S. rubislaw S. goettingen S. guinea S.1,4,5,12:-:1,2 S. cholerae suis S. chester (e,h:e,n,x,z 17) S. dublin (g, p) and S. monschaui (m, t)
1000 x 100x 30x 300X 75 x 500X 200x 300x
1 ml 10 ml 33 ml 3 ml 13 ml 2ml 5ml 3 ml
+ + + +
+
a All H antisera were prepared with the method of Bader and Winkler (1).
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P.A.M.Guinee, W.H.Jansen, and H.M.E.Maas
and kept at 4 dc. Filled trays can be kept at 4 °C for about one week if properly sealed to prevent evaporation for example by stacking.
2.5. Serotyping by means of the mechanized procedure The procedure finally adopted for the screening of Salmonella cultures sent for serotyping is a compromise between optimal sensitivity and maximum efficiency. For example, it was found that the H agglutinations could be read after 21/2 h incubation if the H antigen had a density of about 1 (before staining) and the H antisera were used in a lesser dilution. The shorter incubation time was preferred and the lesser dilution of the antisera had therefore to be accepted. The procedure is as follows. The strains are inoculated onto an agar slant and into 5 ml of home-made broth in 17-18 mm diameter cotton-plugged glass tubes. After overnight incubation at 37°C in a shaking waterbath, one drop of 0.025 microliter 0.4% gentian violet is added to each tube by means of a dropping pipette (Sterilin M35). This live suspension, having an OD600 of about 1 before staining, is used as H antigen. A dropping pipette (Sterilin M36) delivering drops of 50 microliters is placed into each tube, thus being filled without sucking. The volume of 5 ml is not required for the tests but was chosen to avoid the necessity of sucking the live antigen into the pipettes. Drops of each H antigen are added to a row of 8 wells (A-H) in a tray prefilled with 8 different H antisera. The trays are incubated at 45°C in a waterbath on the surface of the water for 21/2 h. The remaining stained bacterial suspension is heated at 100°C for 1 h. After cooling, 10 ml of 0.002% gentian violet solution is added to each. Drops of 50 microliter of each 0 antigen are added to a row of 8 wells (A-H), 6 of which have been prefilled with 6 different 0 antisera. The trays are placed in an incubator at 37°C, piled up and with an empty tray on each stack to prevent evaporation and read after 6-20 h. After reading of the Hand 0 trays, the serotyping is completed by standard manual methods, as far as necessary.
3. Results The mechanized technique by which one antiserum is tested against 120 antigens delivered by means of the multiple dropper was found to be efficient only if a very large number of antisera had to be screened or when antisera were tested which were expected to possess a high specificity. A practical disadvantage of this technique is that the stained antigens in the filling blocks sediment in about 24 hours after which the blocks become useless. The majority of unabsorbed antisera react with many of the standard antigens and must as yet be titrated. The technique by which 15 antisera are tested in 8 decimal dilutions against one antigen per tray was therefore found to be efficient. Moreover this seemingly more laborious technique is a one-step procedure and requires less administrative steps than a two-step procedure. During the development of the mechanized procedure for serotyping, about 2.000 routine strains were serotyped manually as well as with the mechanized procedure. Complete agreement between the results of the two methods was observed. Of the 21.670 Salmonella strains sent for serotyping in 1981, nearly 97% were antigen typed by means of the tray. Only 688 (3.2%) strains did not belong to those 0 groups represented in the tray but to 18 other 0 groups. Of these 18 0 groups, the two groups comprising the largest number of strains were G2 with 259 strains and E4 with 107 strains. About one-third of the 688 O-untypable strains possessed H antigens which were not represented in the tray H antisera. About one-third of the strains had one H phase and the remaining strains had two H phases which could be detected in the tray.
o
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263
Table 3. Typability in 6 0 and 8 H antisera of 21,670 Salm onella strains received for serotyping in 1981 Total - 0 0 - H = 0 - S.
=
21,670
untypable ' typable b untypable and H typable C typhimurium d
fully typed partly typed
= non-So - "E- and G-complex"
typhimurium e
=
remaining fully typed partly typed
100%
688 20,982 1,380 19,602 10,386 4,837
5,549
3.2% 96.8 % 6.4% 90.4% 47.9% 22.3% 25.6%
9,216 5,012
42.5% 23.1%
4,204 954
19.4% 4.4%
3,250
15.0%
a Untypable: the 0 antiserum required is not present in the tray. bAll 0 typable strains were actually typed in the tray. c H typable : one or all antisera required are present in the tray. d S. typhimurium is completely typable. e Mixture of completely and partly typable strains.
Of the 20.982 0 antigen typable strains, 1.380 (6.4%) possessed H antigens which are not represented by the H antisera in the tray. Of the remaining 19.602 strains, 10.386 (47.9%) belonged to S. typhimurium. Of these, 4.837 were completely recognized in the tray and of 5.549 S. typhimurium strains, the second phase was not recognized in the tray . Of the 9.216 0 and H typable, non-So typhimurium strains, 5.012 (23.1 %) were partly typed by means of the "E-" and "G-complex". These results have been summarized in Table 3. They may be considered to be representative for the 4 year period during which the mechanized procedure has been employed.
4. Discussion The mechanized microsystem for serotyping requires a minimum of reagents and has a relatively great output. It was amazing to find that 97% of all strains can be o antigen typed by means of only 6 0 antisera and that such small quantities of monofactor sera are required (see Table 2). A further advantage of the method is that each positive reaction is accompanied by 5 to 7 negative controls. The task of the laboratory worker is considerably relieved and two workers turned out to be able to completely serotype 20.000-25.000 Salmonella cultures per year. The technique has a great degree of versatility. For serotyping, the choice of antisera can easily be adapted to the prevalence of serotypes. When used for the control of antisera, the technique can be adapted to the expected quality of the antisera to be tested.
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References 1. Bader, R.-E. und H. Winkler: Experimentelle Untersuchungen zur Herstellung reiner H-Seren. Zbl. Bakt., 1. Abt. Orig. 195 (1965) 541-543 2. Edwards, P. R. and W. H. Ewing: Identification of Enterobacteriaceae. Burgess, Minneapolis (1962) 3. Guinee, P.A.M., C.M.Agterberg, and W.H.Jansen: Escherichia coli 0 antigen typing by means of a mechanized microtechnique. Appl. Microbiol. 24 (1972) 127-131 4. Guinee, P.A.M., L.Strackee, and W.H.Jansen: An automatic device for the serotyping of Salmonella. Antonie v. Leeuwenhoek 40 (1974) 607-608 5. Kauffmann, F.: Die Bakteriologie der Salmonella-Species. E. Munksgaard, Copenhagen (1961) 6. Schlecht, S. und O. Westphal: Dber die HersteIIung von Antiseren gegen die somatischen (0-) Antigene von Salmonellen. 1. Mitteilung: Untersuchungen tiber Agglutinintiter. Zbl. Bakt., 1. Abt. Orig. 204 (1967) 335-355
Dr. P.A.M.Guinee, Laboratory for Bacteriology, National Institute of Public Health, P. O. Box 1, 3720 BA Bilthoven, The Netherlands
Mechanized Procedures for the Serology of Salmonella Mechanisierte Verfahren fiir die Serologie von Salmonellen P. A. M. GUINEE, W. H. JANSEN, and H. M. E. MAAS Laboratory for Bacteriology, National Institute of Public Health, Bilthoven, The Netherlands
With 1 Figure' Received February 28, 1983
Abstract A mechanized microtechnique originally designed for the serotyping of E. coli was adapted to the specificity control of diagnostic Salmonella agglutinating antisera as well as for the serotyping of Salmonella. Salmonella strains sent for serotyping were inoculated into a tube with broth which was used as H antigen and, after heating also as 0 antigen. The agglutination reactions were carried out in clear plastic trays with U formed wells. The antigens were stained with gentian violet in order to obtain a better contrast. With only 6 monofactor 0 sera, 97% of 100.000 cultures received for serotyping in 1979-1982 could be 0 antigen typed. Eight H antisera were sufficient for the complete serotyping of 27% of these strains whereas 64% was partly typed. The technique has a high degree of versatility.
Zusammenfassung Eine mechanisierte Mikrotechnik, die urspriinglich fiir die Serotypisierung von E. coli entwickelt ist, wurde fiir die Oberwachung der Spezifizitat von diagnostischen Salmonellenagglutinierenden Antiseren und fiir die Serotypisierung von Salmonellen modifiziert. Zur Serotypisierung eingeschickte Salmonellenstamme wurden in ein Rohrchen mit Bouillon geimpft. Nach Inkubation wurde diese Keimsuspension als H-Antigen verwendet und dann - nach Erhitzen - auch als O-Antigen. Die Agglutinationsreaktionen wurden in durchsichtigen Mikrotiterplatten mit U-formigen Vertiefungen ausgefiihrt. Die Antigene wurden mit Gentianaviolett gefarbt, urn einen besseren Kontrast zu erreichen. Mit nur 6 Monofaktor-O-Seren konnten 97% von 100.000 Kulturen, die von 1979-1982 zur Serotypisierung eingegangen sind, O-Antigen typisiert werden . 8 H-Antiseren geniigten zur vollstandigen Serotypisierung von 27% dieser Stamme, wahrend 64% zum Teil typisiert wurden. Das Verfahren ist sehr vielseitig verwendbar.
1. Introduction We have reported previously on attempts to develop an automatic device for serotyping of Salmonella (4). Unfortunately the device did not become operational.
Serology of Salmonella
259
This was mainly due to the fact that bacterial contamination of the reagents in the dispensing needles and tubings could not be sufficiently prevented. On the other hand, the mechanized microtechnique for the 0 antigen typing of E. coli was very successful (3). This technique could also be employed for the specificity control of agglutinating antisera and, in a later stage, for the serotyping of Salmonella. In this paper, we describe the procedures finally adopted for the control of antisera and for serotyping. The results of serotyping 21670 Salmonella strains in 1981 are reported.
2. Materials and Methods 2.1. Buffers, stains and medium Formalinized phosphate-buffered saline (FPBS): 7.2 ml of 35% formalin; NaCl, 2 g; Na2HP04·2H20, 1.26 g; KH 2PO" 0.42 g; distilled water, 1000 ml. Gentian violet stock solution (stock): solution of 1% gentian violet in distilled water. 0.4% gentian violet solution: 4 parts of stock and 6 parts of FPBS. 0.004% gentian violet solution: 4 ml of stock in 1000 ml FPBS. 0.002% gentian violet solution: 2 ml of stock in 1000 ml FPBS. One liter of home-made broth is prepared from fat-free beef, 125 g to which is added peptone (Difco), 2.5g; NaCl, 1.25g; 15% (w/v) solution of Na 2C0 3 ·1·H 2 0, 4ml and NaOH 5 M, 3 ml; pH 7.5. Salmonella strains cultured in home-made broth overnight in a shaking waterbath at 37°C yield an OD 600 of about 1.
2.2. Production of antisera The 0 antisera used were prepared and absorbed with standard methods (2,5) according to the immunization scheme of Schlecht and Westphal (6). 05 antiserum was produced with a formalinized suspension of a non-motile variant of S. paratyphi B (Le Minor, personal communication). The H antisera were prepared with the method described by Bader and Winkler (1).
2.3. Control of antisera The multiple dropper containing 120 syringes as originally described (3) has been replaced by an apparatus commercially built to our specifications. This dropper delivers 0.033 ml instead of 0.037 ml by the original one so that about 55 trays (Conphar 120, Continental Pharma, Zutphen, The Netherlands) can be filled from a filling block. The filling block previously described (3) contained 120 separate reservoirs in 8 (A-H) rows of 15 (1-15) so that 120 different antisera could be dispensed (Fig. 1). The mechanized technique can be employed for the specificity control of antisera in a variety of ways. The extreme possibilities per tray are that one antiserum is tested against 120 different antigens or that 120 antisera are tested against one antigen. An intermediate possibility of titrating 15 antisera against one antigen turned out to be very practical. For the extreme possibility mentioned above, two sets of 120 "standard" antigens were designed comprising all Salmonella 0 and H antigens respectively. The "standard" 0 set comprised all 0 antigens in duplicate, except 054, 55 and 67 which were single, and the 0 antigens of Salmonella 0 group A-G which were in triplicate. The "standard" H set comprised all H antigens and the frequently encountered H antigens in 2- to 4-fold. Each antigen was prepared from a different serovar strain. Unabsorbed antisera were tested in the serum dilutions 1: 50 and 1: 100, absorbed sera in the dilutions 1: 25 and 1: 50. Flagellar antisera were tested against the 0 and H set, 0 antisera only against the 0 set unless the antiserum had been prepared with unheated bacteria, e.g. 05 antiserum prepared with formalinized S. paratyphi B suspension. The optimal density of the antigens was determined by checkerboard titrations
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A
B C D
E F G
H
0 0 0 0 0 0 0 0
2
3
4
5
6
7
8
9
10 11 12 13 14 15
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Fig. 1. Filling block and tray containing 120 reservoirs and cups respectively.
testing dilutions of Hand 0 antigens against decimal dilutions of antisera. All antigens were stained by adding an equal volume of 0.004% gentian violet solution (final concentration 0.002%). H antigens with an OD600 of 0.8 before staining gave strong and high titred agglutinations after 4-5 h incubation at 45°C. Negative reactions are indicated by a sharp point, positive reactions by a carpet as described before (3). Positive H reactions tend to develop a point after prolonged incubation. The optimal density for 0 antigens was 0.4 before staining when incubated at 37°C. The reactions can be read after 6-20 h incubation at 37°C. The trays were filled with 50 microliter of one dilution of the antiserum to be tested using a dropping pipette (one drop = 50 microliter, Sterilin M36). The antigens were delivered by means of the multiple dropper. If necessary, the syringes of the dropper were rinsed with diluted acetic acid to remove the stain followed by rinsing with water. When using the afore-mentioned intermediate possibility for testing antisera, 15 different antisera were titrated against the standard antigens. The antisera were diluted manually (1: 10-1: 1280 in FPBS) and the dilutions applied to the reservoirs A-H of row 1, 2, 3 etc. of one or more filling blocks (Fig. 1). Each standard antigen was added to one tray by means of a dropping pipette. 2.4. Choice and control of antisera to be used in mechanized serotyping The choice of antisera to be used was based on several considerations: (i) the frequency distribution of the various serotypes in The Netherlands; (ii) the form of the tray to be used: 8 strains against 15 antisera or 15 strains against a maximum of 8 antisera; and (iii) the dilution and concomitantly the "price" of the antisera. The most frequently found Table 1. Most frequently found Salmonella serotypes in The Netherlands between 1970 and 1980 typhimurium panama enteritidis infantis S.hadar S. oranienburg S. montevideo S. newport S.agona S. bovis morbificans
S. S. S. S.
1,4, (5), 12 : i : 1,2 1,9,12 : I, v : 1,5 1,9,12 :g,m 6, 7 :r : 1,5 6,8 : zlO : e, n, x 6,7 : m, t 6,7 : g, m, s:6,8 : e, h : 1,2 : f, g, S 4, 12 6,8 : 1,5 :r
Serology of Salmonella
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serotypes in The Netherlands are given in Table 1. Serotypes belonging to 0 group E1 (= 03,10) do not rank in Table 1, but total up to about 4%. Therefore, the following 60 antisera and 8 H antisera are used: 04, OS, 07, 08, 09, 010 and i, r, v, Z10, 2, 5, "E complex" and "G complex". "E-complex" consists of e, hand e, n, x, z17 antiserum and "G complex" stands for the combination of g, p and m, t antiserum. All other antisera are so-called monofactor antisera; they react with only one antigenic determinant. In addition to the control procedures already described, these antisera underwent extra controls. 0 antisera were tested in trays against 10 strains recently received for serotyping (= routine strains) possessing the homologous 0 antigen. The dilution to be used for serotyping was half of the lowest titer found with any of the 10 routine strains and reduced to a manageable dilution. For example, a particular batch of monofactor 07 antiserum gave titres with 10 routine strains ranging from 160-1280. The manageable dilution chosen was 1: 50. As will be described in the next paragraph, the H antigens of strains to be serotyped have a density of about 1 for practical reasons. With such antigens the incubation time could be reduced to 2'/2 h. The titers obtained under these conditions are one or two steps lower. The H antisera to be used for mechanized serotyping were therefore tested also against H antigens prepared from 10 routine strains under the conditions described above. These 10 strains had previously been found to react in such an antiserum in the slide agglutination using the growth in the condens water of an agar slant culture as antigen. The dilution giving optimal reactivity with as many routine cultures as possible was chosen, provided that it was negative with all heterologous standard monophasic H antigens. Details of the antisera as they have been used for mechanized serotyping over 4 years, are given in Table 2. Filling blocks were adapted to dispense 6-8 antisera in rows of 15 by interconnecting each row of wells (1-15). Filling blocks were closed with adhesive tape
Table 2. Antisera used for the mechanized serotyping of Salmonella strains antiserum
produced with
absorbed
dilution used
approximate quantity of undiluted antiserum required annually
04 05 07 08 09 010
S. bredeney 1 h 100 DC S. paratyphi B formalinized S. infantis 1 h 100 DC S. virginia 1 h 100 DC S. goettingen 1 h 100 DC S. muenster 1 h 100 DC
+ + +
150 x 50X 50x 100X 75 x 75 x
7 ml 20ml 20ml 10 ml 13 ml 13 ml
Hi Hr H(v) H z10 H(2) H(5) "E-complex" "G-complex"
S. bonariensis' S. rubislaw S. goettingen S. guinea S.1,4,5,12:-:1,2 S. cholerae suis S. chester (e,h:e,n,x,z 17) S. dublin (g, p) and S. monschaui (m, t)
1000 x 100x 30x 300X 75 x 500X 200x 300x
1 ml 10 ml 33 ml 3 ml 13 ml 2ml 5ml 3 ml
+ + + +
+
a All H antisera were prepared with the method of Bader and Winkler (1).
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and kept at 4 dc. Filled trays can be kept at 4 °C for about one week if properly sealed to prevent evaporation for example by stacking.
2.5. Serotyping by means of the mechanized procedure The procedure finally adopted for the screening of Salmonella cultures sent for serotyping is a compromise between optimal sensitivity and maximum efficiency. For example, it was found that the H agglutinations could be read after 21/2 h incubation if the H antigen had a density of about 1 (before staining) and the H antisera were used in a lesser dilution. The shorter incubation time was preferred and the lesser dilution of the antisera had therefore to be accepted. The procedure is as follows. The strains are inoculated onto an agar slant and into 5 ml of home-made broth in 17-18 mm diameter cotton-plugged glass tubes. After overnight incubation at 37°C in a shaking waterbath, one drop of 0.025 microliter 0.4% gentian violet is added to each tube by means of a dropping pipette (Sterilin M35). This live suspension, having an OD600 of about 1 before staining, is used as H antigen. A dropping pipette (Sterilin M36) delivering drops of 50 microliters is placed into each tube, thus being filled without sucking. The volume of 5 ml is not required for the tests but was chosen to avoid the necessity of sucking the live antigen into the pipettes. Drops of each H antigen are added to a row of 8 wells (A-H) in a tray prefilled with 8 different H antisera. The trays are incubated at 45°C in a waterbath on the surface of the water for 21/2 h. The remaining stained bacterial suspension is heated at 100°C for 1 h. After cooling, 10 ml of 0.002% gentian violet solution is added to each. Drops of 50 microliter of each 0 antigen are added to a row of 8 wells (A-H), 6 of which have been prefilled with 6 different 0 antisera. The trays are placed in an incubator at 37°C, piled up and with an empty tray on each stack to prevent evaporation and read after 6-20 h. After reading of the Hand 0 trays, the serotyping is completed by standard manual methods, as far as necessary.
3. Results The mechanized technique by which one antiserum is tested against 120 antigens delivered by means of the multiple dropper was found to be efficient only if a very large number of antisera had to be screened or when antisera were tested which were expected to possess a high specificity. A practical disadvantage of this technique is that the stained antigens in the filling blocks sediment in about 24 hours after which the blocks become useless. The majority of unabsorbed antisera react with many of the standard antigens and must as yet be titrated. The technique by which 15 antisera are tested in 8 decimal dilutions against one antigen per tray was therefore found to be efficient. Moreover this seemingly more laborious technique is a one-step procedure and requires less administrative steps than a two-step procedure. During the development of the mechanized procedure for serotyping, about 2.000 routine strains were serotyped manually as well as with the mechanized procedure. Complete agreement between the results of the two methods was observed. Of the 21.670 Salmonella strains sent for serotyping in 1981, nearly 97% were antigen typed by means of the tray. Only 688 (3.2%) strains did not belong to those 0 groups represented in the tray but to 18 other 0 groups. Of these 18 0 groups, the two groups comprising the largest number of strains were G2 with 259 strains and E4 with 107 strains. About one-third of the 688 O-untypable strains possessed H antigens which were not represented in the tray H antisera. About one-third of the strains had one H phase and the remaining strains had two H phases which could be detected in the tray.
o
Serology of Salmonella
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Table 3. Typability in 6 0 and 8 H antisera of 21,670 Salm onella strains received for serotyping in 1981 Total - 0 0 - H = 0 - S.
=
21,670
untypable ' typable b untypable and H typable C typhimurium d
fully typed partly typed
= non-So - "E- and G-complex"
typhimurium e
=
remaining fully typed partly typed
100%
688 20,982 1,380 19,602 10,386 4,837
5,549
3.2% 96.8 % 6.4% 90.4% 47.9% 22.3% 25.6%
9,216 5,012
42.5% 23.1%
4,204 954
19.4% 4.4%
3,250
15.0%
a Untypable: the 0 antiserum required is not present in the tray. bAll 0 typable strains were actually typed in the tray. c H typable : one or all antisera required are present in the tray. d S. typhimurium is completely typable. e Mixture of completely and partly typable strains.
Of the 20.982 0 antigen typable strains, 1.380 (6.4%) possessed H antigens which are not represented by the H antisera in the tray. Of the remaining 19.602 strains, 10.386 (47.9%) belonged to S. typhimurium. Of these, 4.837 were completely recognized in the tray and of 5.549 S. typhimurium strains, the second phase was not recognized in the tray . Of the 9.216 0 and H typable, non-So typhimurium strains, 5.012 (23.1 %) were partly typed by means of the "E-" and "G-complex". These results have been summarized in Table 3. They may be considered to be representative for the 4 year period during which the mechanized procedure has been employed.
4. Discussion The mechanized microsystem for serotyping requires a minimum of reagents and has a relatively great output. It was amazing to find that 97% of all strains can be o antigen typed by means of only 6 0 antisera and that such small quantities of monofactor sera are required (see Table 2). A further advantage of the method is that each positive reaction is accompanied by 5 to 7 negative controls. The task of the laboratory worker is considerably relieved and two workers turned out to be able to completely serotype 20.000-25.000 Salmonella cultures per year. The technique has a great degree of versatility. For serotyping, the choice of antisera can easily be adapted to the prevalence of serotypes. When used for the control of antisera, the technique can be adapted to the expected quality of the antisera to be tested.
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References 1. Bader, R.-E. und H. Winkler: Experimentelle Untersuchungen zur Herstellung reiner H-Seren. Zbl. Bakt., 1. Abt. Orig. 195 (1965) 541-543 2. Edwards, P. R. and W. H. Ewing: Identification of Enterobacteriaceae. Burgess, Minneapolis (1962) 3. Guinee, P.A.M., C.M.Agterberg, and W.H.Jansen: Escherichia coli 0 antigen typing by means of a mechanized microtechnique. Appl. Microbiol. 24 (1972) 127-131 4. Guinee, P.A.M., L.Strackee, and W.H.Jansen: An automatic device for the serotyping of Salmonella. Antonie v. Leeuwenhoek 40 (1974) 607-608 5. Kauffmann, F.: Die Bakteriologie der Salmonella-Species. E. Munksgaard, Copenhagen (1961) 6. Schlecht, S. und O. Westphal: Dber die HersteIIung von Antiseren gegen die somatischen (0-) Antigene von Salmonellen. 1. Mitteilung: Untersuchungen tiber Agglutinintiter. Zbl. Bakt., 1. Abt. Orig. 204 (1967) 335-355
Dr. P.A.M.Guinee, Laboratory for Bacteriology, National Institute of Public Health, P. O. Box 1, 3720 BA Bilthoven, The Netherlands