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Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines
FEMS Microbiology Letters 192 (2000) 101^106
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Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines Cyril Barbezange, Gwennola Ermel *, Cathy Ragimbeau, Florence Humbert, Gilles Salvat AFSSA-Ploufragan, Unit HQPAP, P.O. Box 53, Zoopoªle, 22440 Ploufragan, France Received 2 August 2000; accepted 30 August 2000
Abstract Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of M3985 , a genetically modified organism, were variants and showed different macrorestriction profiles. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Macrorestriction genotyping; Vaccine; Salmonella typhimurium
1. Introduction In France, the serovars enteritidis and typhimurium of Salmonella enterica subsp. enterica (S. enteritidis and S. typhimurium), are responsible for more than one half of food-poisoning in humans and are mainly associated with poultry products [1]. The prevention against salmonellosis is di¤cult because of the high persistence of Salmonella in the environment of breeders, layers and broilers [2^4], the oral^faecal way of contamination [5,6] and the large range of chronic carrier hosts [7,8]. According to the European Directives 92/117/EEC and 97/22/EEC, France has planned eradication of S. enteritidis and S. typhimurium by slaughtering contaminated breeding £ocks for layers and broilers [9,10]. Among alternative methods, vaccines are attractive [11^ 13]. Avirulent live vaccines are considered as more e¤cient to prevent the spread of Salmonella contamination in the poultry process than attenuated or particular vaccines [14,15]. A live Salmonella vaccine strain should be completely avirulent for both men and animals, highly immunogenic ^
* Corresponding author. Tel. : +33 (2) 96 016 287; Fax: +33 (2) 96 016 283 ; E-mail : [email protected]
immunity persistence during the whole animal life, prevention of the infection of deep organs and reduction of the colonization of the digestive tract, and thus for di¡erent serovars of Salmonella, and genetically stable. Their own attenuated phenotype must be host- and food-independent. Culture, conservation and administration are also important criteria [16]. During a study on the environmental persistence and the spread in organs of chickens, several isolates of three live vaccine strains of S. typhimurium were collected in order to analyze their genetic stability. The isolates of each vaccine strain were collected at di¡erent times after inoculation to chicks, and DNA stability was checked by macrorestriction using two enzymes (SpeI and XbaI) followed by pulsed ¢eld gel electrophoresis (PFGE). 2. Materials and methods 2.1. Bacterial isolates and growth conditions Of the 117 Salmonella typhimurium isolates used in this study (Table 1), 42 came from di¡erent chicken organs between 72 h to 10 days after a single oral administration of three live vaccine strains (vacT, Zoosaloral, M3985 ) and of their parental ones (M415 for vacT and Zoosaloral,
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 1 6 - X
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M3761 for M3985 ) to individually reared 1-day-old chicks (assay 1). The remaining 75 S. typhimurium isolates were obtained from the environment and di¡erent chicken organs between 2 and 6 weeks after a single oral administration of the three live vaccine strains to 1-day-old chicks reared in industrial conditions (assay 2). 2.2. Phage typing The Salmonella cultures were grown at 37³C in 5 ml of tryptic soy broth for 6 h. A small quantity of each culture was plated onto nutrient agar (Difco) and allowed to dry at room temperature. One drop of each phage lysate, coming from the Laboratory for Enteric Pathogens (Colindale, London, UK) [17], was spotted onto the inoculated plate and incubation was performed overnight at 37³C. The patterns were determined by viewing clear isolated or con£uent lysis plaques. 2.3. DNA preparation for PFGE The bacterial lawn obtained from an overnight culture on tryptic soy agar was suspended in 2.5 ml of Tris^NaCl (TN) solution (0.01 mol l31 Tris^HCl, 1 mol l31 NaCl, pH 7.6). Cells were harvested and washed twice with 2 ml of TN. Agarose plugs (100 Wl) were prepared by mixing 50 Wl of adjusted cell suspension (OD600 nm = 1.5) to 50 Wl of 1% (w/ v) agarose (Agarose Standard, Eurobio, France). The solidi¢ed agarose plugs were then incubated in ESP lysis bu¡er (0.5 mol l31 EDTA, pH 9, 1% (w/v) lauroyl sarcosine, 1 mg ml31 proteinase K) at 50³C at least for 40 h. After two washes of 30 min at room temperature with TE bu¡er (0.01 mol l31 Tris^HCl, 0.001 mol l31 Na2 EDTA, pH 8), the proteinase K was inactivated by incubating agarose plugs in 2 mmol l31 aminoethyl-benzenesulfonyl £uoride (PEFABLOC0 , Boehringer Mannheim, Germany). Then, plugs were washed twice with TE bu¡er as previously described and were cut in four thin slices. 2.4. Restriction endonuclease digestion and PFGE conditions A quarter of plug was used for restriction endonuclease digestions in separate reaction with 40 U of either SpeI or XbaI (Boehringer) under the manufacturer's recommended conditions, in a ¢nal volume of 100 Wl and for 5 h of incubation at the appropriate temperature. The separation of the restricted fragments was performed by PFGE using the CHEF-DRIII system (BioRad Laboratories, USA). Agarose gels (1%) prepared in 0.5U TBE (Tris 45 mmol l31 , boric acid 45 mmol l31 , EDTA 1 mmol l31 ) were subjected to electrophoresis for 22 h at 220 V, at 14³C, with ramped pulse times from 20 to 40 s for the ¢rst 12 h and from 7 to 13 s for the last 10 h.
2.5. Analysis of the electrophoresis patterns Agarose gels were stained in a solution of ethidium bromide (0.5 Wg ml31 ) during 30 min. Images were captured under UV illumination by a video system (gel DOC 1000 system, Bio-Rad) and visual analysis of the electrophoretic patterns was performed. 3. Results 3.1. Analysis using macrorestriction with SpeI and XbaI The di¡erent pro¢les are shown in Fig. 1 and are noticed for all the collected isolates in Table 1. The two strains M3761 and M3985 were only di¡erentiated by macrorestriction with XbaI (Fig. 1, lanes 3 and 5). A shift of a 200-kbp DNA-fragment allowed the two di¡erent patterns X1 and X2. The isolate 1607 derived from M3761 (from assay 1) showed a modi¢ed pro¢le X1a (Fig. 1, lane 2), due to an additional fragment, the size of which is approximately 90 kbp. For the M3985 vaccine strain, nine variant isolates were observed, corresponding to six combined pro¢les using the two restriction enzymes (e.g. S1aX2). Indeed, macrorestriction using XbaI showed ¢ve di¡erent patterns (Fig. 1, lanes 6^10). The patterns X2a^d di¡ered from X2 (M3985 pro¢le) by a shift-up of the 450kbp restriction fragment (Fig. 1, lanes 6^9), and for X2e, this fragment vanished (Fig. 1, lane 10). Moreover, the pro¢les X2c^e possessed modi¢ed DNA fragments ranging from 220 to 240 kbp (Fig. 1, lanes 7, 9 and 10). The macrorestriction using SpeI gave three di¡erent pro¢les (S1a^c) which di¡ered from the M3985 pattern S1 (Fig. 1, lanes 21^24). The pro¢le S1a showed an additional restriction fragment (about 190 kbp) and a lower fragment of 390 kbp instead of the 420-kbp fragment present in all the other pro¢les (Fig. 1, lane 21). The pro¢les S1b and S1c were characterized by the absence of the 190-kbp restriction fragment : an additional 320-kbp fragment allowed the distinction of pro¢le S1c (Fig. 1, lanes 22 and 24). From the three strains, vaccine ones (vacT and Zoosaloral) and parental one (M415), only the Zoosaloral was distinguished by the use of SpeI. Indeed, pro¢le S3 of Zoosaloral di¡ered from pro¢le S2 of both vacT and M415 by an additional fragment of approximately 340 kbp (Fig. 1, lanes 15 and 16). No variant isolates were found from experiments where either M415 or vacT were assayed. On the other hand, two variants (isolates 1244 and 1417, Table 1) were obtained after passage of Zoosaloral through animals. The variant isolate 1244 was only visualized using SpeI-macrorestriction and had the pro¢le S3a which showed an additional band at 195 kbp and a lower fragment of 390 kbp instead of the 420-kbp fragment present in pro¢le S3 of Zoosaloral (Fig. 1, lane 17). The variant isolate 1417 had di¡erent pro¢les (S3b and X3a) with both restriction enzymes SpeI and XbaI.
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Fig. 1. Composite numerical photograph of macrorestriction pro¢les obtained using either XbaI (lanes 1^13) or SpeI (lanes 14^24). Lanes 1, 4, 11, 14, 18 and 20 correspond to the molecular standard weight V ladder (NEN Biolabs). Lanes 2, 3, 5, 6, 7, 8, 9, 10, 12 and 13 correspond respectively to XbaI pro¢les: X1a, X1, X2, X2a, X2c, X2b, X2d, X2e, X3a and X3. Lanes 15, 16, 17, 19, 21, 22, 23 and 24 correspond respectively to SpeI pro¢les : S2, S3, S3a, S3b, S1a, S1b, S1 and S1c.
The pro¢le S3b is distinguished from S3 by the vanishing of two restriction fragments (220 and 225 kbp) and apparition of two new fragments (150 and 290 kbp) (Fig. 1, lane 19). In the pro¢le X3a, two extra bands of 340 and 500 kbp were observed (Fig. 1, lane 12). 3.2. Phage typing Phage typing was only realized on all variant isolates in order to ensure that they derived from the original vaccine or parental strains. The isolate 1607 and the nine variant isolates of M3985 had the same phagetype, DT191, identical to those of M3761 and M3985 (Table 1). Both Zoosaloral variants (1244 and 1417), the vaccine strains (Zoosaloral and vacT) and the parental strain M415 had the same phagetype DT009 (Table 1). 4. Discussion Molecular typing methods provide useful tools for an exact and reliable di¡erentiation of Salmonella vaccine strains and are helpful to check the genetic stability of live attenuated vaccines used to prevent colonization of
chickens. In the present study, two macrorestriction techniques were used to characterize three S. typhimurium vaccine strains, the parental strains and the collected isolates obtained after their passage through chickens. Genotypic analysis, by the resolution of restriction fragments of genomic DNA upon PFGE, showed that the vaccine strain M3985 is the most genetically unstable. Indeed, nine variants on 32 collected isolates were detected using SpeI or/and XbaI macrorestrictions. Di¡erences between restriction patterns were observed and implied that mutations highly occurred in vaccine strain M3985 . It is noticeable that this strain is a genetically modi¢ed organism resulting from two directed deletions (vcya vcrp) [18]. Therefore, this could a¡ect its genetic stability. However, one variant out of eight isolates collected from the wild parental strain M3761 was also observed. This phenomenon showed that the wild strain may also present some genetic instability, and might induce the instability of the derived attenuated vaccine strain M3985 . The impact of the directed deletions could also be signi¢cant. A certain genetic instability was noticed in the vaccine strain Zoosaloral: two variants out of 51 collected isolates. This result di¡ers from those obtained by Schwarz and Liebisch [19]. Using XbaI- and SpeI-macrorestriction, these authors did not observe
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Table 1 Isolates from the ¢ve Salmonella strains used for the study of genetic stability Source
Isolate
Assay
M415 845, 853, 861 871 927 1143
1 1 1 1
668, 676, 686, 695, 704, 714 622 891, 893 895, 897 899 901 1359, 1364, 1369, 1374, 1381, 1436 1438
1 2 2 2 2 2 2 2
1255, 1273, 1291 1345 1302, 1312, 1318, 1328, 1334, 1343, 1346 616, 630 883, 885, 903, 905 887, 907 889, 909 911 1218, 1220, 1228, 1230 1222, 1224, 1232, 1234 1226, 1236 1244 1246 1384, 1390 1348, 1350, 1352, 1355, 1421, 1425, 1429, 1433, 1387, 1393, 1439, 1443, 1444, 1446 1417
1 1 1 2 2 2 2 2 2 2 2 2 2 2 2
1615, 1625, 1633, 1643 1607 1723, 1731, 1739
1 1 1
1933, 1962 1946, 1954, 1968 1938, 1961 1976, 1980, 1984, 1988 638 913, 914 915 916 917 918, 919 921 1238, 1240 1241 1249 1400, 1407, 1450, 1456, 1469 1413 1463 1461
1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2
vacT
Zoosaloral
M3761
M3985
2
Sample
control caeca liver caeca caeca control caeca swab ground wall ¢lter swab caeca liver control caeca jejunum caeca swab ground wall ¢lter swab ground wall ¢lter swab swab jejunum caeca caeca control caeca caeca caeca control jejunum caeca liver liver swab ground wall wall ¢lter swab swab ground ¢lter swab caeca caeca caeca jejunum
Day of sampling
D0+72 h D0+72 h D0+6 d D0+10 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+6 w D0+6 w D0+6 d D0+10 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+5 w D0+5 w D0+5 w D0+5 w D0+5 w D0+6 w D0+6 w D0+6 w D0+6 d D0+6 d D0+10 d D0+6 d D0+6 d D0+6 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+4 w D0+4 w D0+5 w D0+5 w D0+5 w D0+6 w D0+6 w D0+6 w D0+6 w
Time of sampling is given as hours (h), days (d) or weeks (w) from the day of inoculation (D0) nd: not determined
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Pattern
Phage type
SpeI
XbaI
S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3a S3 S3 S3
X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3
S3b S1 nd nd nd S1 nd nd nd nd S1a S1 S1b S1b S1b S1 S1b S1 S1 S1b S1 S1b S1c S1c
X3a X1 X1 X1a X1 X2 X2 X2 X2 X2 X2 X2 X2a X2b X2b X2 X2c X2 X2 X2b X2 X2d X2e X2e
DT009
DT009
DT009
DT009
DT009 DT191 DT191 DT191
DT191 DT191 DT191 DT191 DT191
DT191 DT191 DT191 DT191
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modi¢ed pro¢les in seven isolates of Zoosaloral collected over a 22-month period. They concluded that the Zoosaloral strain was genetically stable. It is quite interesting to note that the use of XbaI enzyme was helpful for us to detect a variant isolate of Zoosaloral. However, for Frech et al. [20], SpeI enzyme was of more interest than XbaI or BlnI to distinguish Zoosaloral strain from ¢eld DT009 strains of S. typhimurium. VacT is the only vaccine strain studied for which we did not detect variant isolates. A di¡erence between patterns of an original strain and its variants corresponds to a change in genome, so to a mutation. These variations concern only few DNA fragments, and the general pro¢le suits the original pattern: there is evidence for genomic relationship between the variants and the original strain. Moreover, all the tested isolates present the classical biochemical properties of their original vaccine strain (classical criteria of the Salmonella genus for M3761 , M415, vacT and Zoosaloral, but H2 S2 negative for M3985 ). Phage typing con¢rmed that variant isolates came from the inoculated strain. It would be then interesting to complete those results with the use of other enzymes or even other molecular tools that seem helpful to detect genomic variations among S. typhimurium strains, as IS200 typing [21]. We noticed that patterns of variant isolates were never similar to the wild strain ones (when distinguishable), so no reverse mutations were observed. Nevertheless, the origin of the mutations is unknown. It could be punctual mutations, or deletions, or even recombination with other bacterial species from the gut. It could be also mutations independent of passage on animals but connected to the long bacteriological method used for the isolation of Salmonella (in four steps, ISO 6579 :1993). The three vaccine strains are now marketed. M3985 vaccine strain is commercially available in the United States as Foodsafe 10 (Megan, USA). The other two vaccine strains are commercially available in Germany: vacT by TAD Pharmazeutisches Werk GmbH (Cuxhaven, Germany) and Zoosaloral by Impfsto¡werk Dessau-Tornau GmbH (RoMlau, Germany). Curtiss and co-workers have well studied the e¤ciency of M3985 vaccine strain [13,22]. They proved the e¤ciency of this vaccine strain to prevent colonization of the gut and to protect chicken after challenge with wild strains. On the contrary, the two other vaccine strains, vacT and Zoosaloral, appeared to be poorly inhibitory against infection with wild strains, even if they were shown to be immunogenic [23]. We were able to detect variations in the genetic stability of those vaccine strains of S. typhimurium, but we were unable to link them to a potential impact on safety or even e¤ciency. What are the real properties of these variant isolates with regards to safety, stability and e¤ciency? Would they not be better candidates for use as vaccines? We have also to keep in mind that the use of such live vaccine strains in an animal production process might
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generate hygiene and security problems for products intended to human alimentation.
References [1] Haeghebaert, S., Delarocque Astagneau, E. and Vaillant, V. (1999) Les toxi-Infections Alimentaires Collectives en France en 1997. Annual Epidemiological Report. Infectious Diseases Epidemiology in France in 1997. Re¨seau National de Sante¨ Publique, Saint-Maurice, France. [2] Bailey, J.S. (1993) Control of Salmonella and Campylobacter in poultry production, a summary of work at Russell Research Centre. Poult. Sci. 72, 1169^1173. [3] Bale, J., Bennet, P.M., Beringer, J.E. and Hinton, M.H. (1993) The survival of bacteria exposed to desiccation on surfaces associated with farm buildings. J. Appl. Bacteriol. 75, 519^528. [4] Davies, R.H. and Wray, C. (1996) Persistence of Salmonella enteritidis in poultry units and poultry food. Br. Poult. Sci. 37, 589^ 596. [5] Lahellec, C. and Colin, P. (1985) Relationship between serotypes of Salmonella from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult. Sci. 26, 179^186. [6] Cox, N.A., Bailey, J.S. and Berrang, M.E. (1996) Alternative routes for Salmonella intestinal tract colonization of chicks. J. Appl. Poult. Res. 5, 282^288. [7] Henzler, D.J. and Opitz, H.M. (1992) The role of mice in the epizootiology of Salmonella enteritidis infection in chicken layer farms. Avian Dis. 36, 625^631. [8] Wall, R.G., Davies, S., Threfall, E.J., Ward, L.R. and Ewbank, A.J. (1995) Chronic carriage of multidrug resistant Salmonella typhimurium in a cat. J. Small Anim. Pract. 36, 279^281. [9] Anonymous (1998) Arreªte¨ du 26 octobre 1998 relatif a© la lutte contre les infections a© Salmonella enteritidis ou Salmonella typhimurium dans les troupeaux de reproduction de l'espe©ce Gallus gallus en ¢lie©re chair. JORF du 8.9.98. [10] Anonymous (1998) Arreªte¨ du 26 octobre 1998 relatif a© la lutte contre les infections a© Salmonella enteritidis ou Salmonella typhimurium dans les troupeaux de l'espe©ce Gallus gallus en ¢lie©re ponte d'Eufs de consommation. JORF du 8.9.98. [11] Barrow, P.A., Lovell, M.A. and Berchieri Jr., A. (1991) The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella enteritidis phage type 4. Avian Path. 20, 681^692. [12] Cooper, G.L., Vanables, L.M., Woodward, M.J. and Hormaeche, C.E. (1994) Vaccination of chickens with strain CVL30, a genetically de¢ned Salmonella enteritidis aroA live oral vaccine candidate. Infect. Immun. 62, 4747^4754. [13] Hassan, J.O. and Curtiss III, R. (1994) Development and evalution of an experimental vaccination program using a live avirulent Salmonella typhimurium strain to protect immunized chickens against challenge with homologous and heterologous Salmonella serotypes. Infect. Immun. 62, 5519^5527. [14] Nassar, T.J., Al-Nakhli, H.M. and Al-Ogaily, Z.H. (1994) Use of live and inactivated Salmonella enteritidis phage type 4 vaccines to immunise laying hens against experimental infection. Rev. Sci Tech. O¡. Int. Epizoot. 13, 855^867. [15] Suphabphant, W., York, M.D. and Pomeroy, B.S. (1982) Use of two vaccines (live G30D or killed RW16) in the prevention of Salmonella typhimurium infections in chickens. Avian Dis. 27, 602^615. [16] Curtiss III, R., Kelly, S.M. and Hassan, J.O. (1993) Live oral avirulent Salmonella vaccines. Vet. Microbiol. 37, 397^405. [17] Anderson, E.S., Ward, L.R. and DeSaxe, J.D.H. (1977) Bacteriophage-typing designations of Salmonella typhimurium. J. Hyg. 78, 297^300. [18] Curtiss III, R. and Kelly, S.M. (1987) Salmonella typhimurium dele-
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tion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect. Immun. 55, 3035^ 3043. [19] Schwarz, S. and Liebisch, B. (1994) Pulsed-¢eld gel electrophoretic identi¢cation of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H. Lett. Appl. Microbiol. 19, 469^472. [20] Frech, G., Weide-Botjes, M., Nussbeck, E., Rabsch, W. and Schwarz, S. (1998) Molecular characterization of Salmonella enterica subsp. enterica serovar Typhimurium DT009 isolates di¡erentiation of the live vaccine strain Zoosaloral from ¢eld isolates. FEMS Microbiol. Lett. 167, 263^269. [21] Schwarz, S. and Liebisch, B. (1994) Use of ribotyping, IS200 typing
and plasmid analysis for the identi¢cation of Salmonella enterica subsp. enterica Serovar Typhimurium vaccine strain Zoosaloral H and its di¡erentiation from wild type strains of the same serovar. Zent. bl. Bakt. 281, 442^450. [22] Curtiss III, R. and Hassan, J.O. (1996) Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet. Immunol. Immunopathol. 54, 365^372. [23] Methner, U., Barrow, P.A., Martin, G. and Meyer, H. (1997) Comparative study of the protective e¡ect against Salmonella colonisation in newly hatched SPF chickens using live, attenuated Salmonella vaccine strains, wild-type Salmonella strains or a competitive exclusion product. Int. J. Food Microbiol. 35, 223^230.
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Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines Cyril Barbezange, Gwennola Ermel *, Cathy Ragimbeau, Florence Humbert, Gilles Salvat AFSSA-Ploufragan, Unit HQPAP, P.O. Box 53, Zoopoªle, 22440 Ploufragan, France Received 2 August 2000; accepted 30 August 2000
Abstract Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of M3985 , a genetically modified organism, were variants and showed different macrorestriction profiles. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Macrorestriction genotyping; Vaccine; Salmonella typhimurium
1. Introduction In France, the serovars enteritidis and typhimurium of Salmonella enterica subsp. enterica (S. enteritidis and S. typhimurium), are responsible for more than one half of food-poisoning in humans and are mainly associated with poultry products [1]. The prevention against salmonellosis is di¤cult because of the high persistence of Salmonella in the environment of breeders, layers and broilers [2^4], the oral^faecal way of contamination [5,6] and the large range of chronic carrier hosts [7,8]. According to the European Directives 92/117/EEC and 97/22/EEC, France has planned eradication of S. enteritidis and S. typhimurium by slaughtering contaminated breeding £ocks for layers and broilers [9,10]. Among alternative methods, vaccines are attractive [11^ 13]. Avirulent live vaccines are considered as more e¤cient to prevent the spread of Salmonella contamination in the poultry process than attenuated or particular vaccines [14,15]. A live Salmonella vaccine strain should be completely avirulent for both men and animals, highly immunogenic ^
* Corresponding author. Tel. : +33 (2) 96 016 287; Fax: +33 (2) 96 016 283 ; E-mail : [email protected]
immunity persistence during the whole animal life, prevention of the infection of deep organs and reduction of the colonization of the digestive tract, and thus for di¡erent serovars of Salmonella, and genetically stable. Their own attenuated phenotype must be host- and food-independent. Culture, conservation and administration are also important criteria [16]. During a study on the environmental persistence and the spread in organs of chickens, several isolates of three live vaccine strains of S. typhimurium were collected in order to analyze their genetic stability. The isolates of each vaccine strain were collected at di¡erent times after inoculation to chicks, and DNA stability was checked by macrorestriction using two enzymes (SpeI and XbaI) followed by pulsed ¢eld gel electrophoresis (PFGE). 2. Materials and methods 2.1. Bacterial isolates and growth conditions Of the 117 Salmonella typhimurium isolates used in this study (Table 1), 42 came from di¡erent chicken organs between 72 h to 10 days after a single oral administration of three live vaccine strains (vacT, Zoosaloral, M3985 ) and of their parental ones (M415 for vacT and Zoosaloral,
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 1 6 - X
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M3761 for M3985 ) to individually reared 1-day-old chicks (assay 1). The remaining 75 S. typhimurium isolates were obtained from the environment and di¡erent chicken organs between 2 and 6 weeks after a single oral administration of the three live vaccine strains to 1-day-old chicks reared in industrial conditions (assay 2). 2.2. Phage typing The Salmonella cultures were grown at 37³C in 5 ml of tryptic soy broth for 6 h. A small quantity of each culture was plated onto nutrient agar (Difco) and allowed to dry at room temperature. One drop of each phage lysate, coming from the Laboratory for Enteric Pathogens (Colindale, London, UK) [17], was spotted onto the inoculated plate and incubation was performed overnight at 37³C. The patterns were determined by viewing clear isolated or con£uent lysis plaques. 2.3. DNA preparation for PFGE The bacterial lawn obtained from an overnight culture on tryptic soy agar was suspended in 2.5 ml of Tris^NaCl (TN) solution (0.01 mol l31 Tris^HCl, 1 mol l31 NaCl, pH 7.6). Cells were harvested and washed twice with 2 ml of TN. Agarose plugs (100 Wl) were prepared by mixing 50 Wl of adjusted cell suspension (OD600 nm = 1.5) to 50 Wl of 1% (w/ v) agarose (Agarose Standard, Eurobio, France). The solidi¢ed agarose plugs were then incubated in ESP lysis bu¡er (0.5 mol l31 EDTA, pH 9, 1% (w/v) lauroyl sarcosine, 1 mg ml31 proteinase K) at 50³C at least for 40 h. After two washes of 30 min at room temperature with TE bu¡er (0.01 mol l31 Tris^HCl, 0.001 mol l31 Na2 EDTA, pH 8), the proteinase K was inactivated by incubating agarose plugs in 2 mmol l31 aminoethyl-benzenesulfonyl £uoride (PEFABLOC0 , Boehringer Mannheim, Germany). Then, plugs were washed twice with TE bu¡er as previously described and were cut in four thin slices. 2.4. Restriction endonuclease digestion and PFGE conditions A quarter of plug was used for restriction endonuclease digestions in separate reaction with 40 U of either SpeI or XbaI (Boehringer) under the manufacturer's recommended conditions, in a ¢nal volume of 100 Wl and for 5 h of incubation at the appropriate temperature. The separation of the restricted fragments was performed by PFGE using the CHEF-DRIII system (BioRad Laboratories, USA). Agarose gels (1%) prepared in 0.5U TBE (Tris 45 mmol l31 , boric acid 45 mmol l31 , EDTA 1 mmol l31 ) were subjected to electrophoresis for 22 h at 220 V, at 14³C, with ramped pulse times from 20 to 40 s for the ¢rst 12 h and from 7 to 13 s for the last 10 h.
2.5. Analysis of the electrophoresis patterns Agarose gels were stained in a solution of ethidium bromide (0.5 Wg ml31 ) during 30 min. Images were captured under UV illumination by a video system (gel DOC 1000 system, Bio-Rad) and visual analysis of the electrophoretic patterns was performed. 3. Results 3.1. Analysis using macrorestriction with SpeI and XbaI The di¡erent pro¢les are shown in Fig. 1 and are noticed for all the collected isolates in Table 1. The two strains M3761 and M3985 were only di¡erentiated by macrorestriction with XbaI (Fig. 1, lanes 3 and 5). A shift of a 200-kbp DNA-fragment allowed the two di¡erent patterns X1 and X2. The isolate 1607 derived from M3761 (from assay 1) showed a modi¢ed pro¢le X1a (Fig. 1, lane 2), due to an additional fragment, the size of which is approximately 90 kbp. For the M3985 vaccine strain, nine variant isolates were observed, corresponding to six combined pro¢les using the two restriction enzymes (e.g. S1aX2). Indeed, macrorestriction using XbaI showed ¢ve di¡erent patterns (Fig. 1, lanes 6^10). The patterns X2a^d di¡ered from X2 (M3985 pro¢le) by a shift-up of the 450kbp restriction fragment (Fig. 1, lanes 6^9), and for X2e, this fragment vanished (Fig. 1, lane 10). Moreover, the pro¢les X2c^e possessed modi¢ed DNA fragments ranging from 220 to 240 kbp (Fig. 1, lanes 7, 9 and 10). The macrorestriction using SpeI gave three di¡erent pro¢les (S1a^c) which di¡ered from the M3985 pattern S1 (Fig. 1, lanes 21^24). The pro¢le S1a showed an additional restriction fragment (about 190 kbp) and a lower fragment of 390 kbp instead of the 420-kbp fragment present in all the other pro¢les (Fig. 1, lane 21). The pro¢les S1b and S1c were characterized by the absence of the 190-kbp restriction fragment : an additional 320-kbp fragment allowed the distinction of pro¢le S1c (Fig. 1, lanes 22 and 24). From the three strains, vaccine ones (vacT and Zoosaloral) and parental one (M415), only the Zoosaloral was distinguished by the use of SpeI. Indeed, pro¢le S3 of Zoosaloral di¡ered from pro¢le S2 of both vacT and M415 by an additional fragment of approximately 340 kbp (Fig. 1, lanes 15 and 16). No variant isolates were found from experiments where either M415 or vacT were assayed. On the other hand, two variants (isolates 1244 and 1417, Table 1) were obtained after passage of Zoosaloral through animals. The variant isolate 1244 was only visualized using SpeI-macrorestriction and had the pro¢le S3a which showed an additional band at 195 kbp and a lower fragment of 390 kbp instead of the 420-kbp fragment present in pro¢le S3 of Zoosaloral (Fig. 1, lane 17). The variant isolate 1417 had di¡erent pro¢les (S3b and X3a) with both restriction enzymes SpeI and XbaI.
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Fig. 1. Composite numerical photograph of macrorestriction pro¢les obtained using either XbaI (lanes 1^13) or SpeI (lanes 14^24). Lanes 1, 4, 11, 14, 18 and 20 correspond to the molecular standard weight V ladder (NEN Biolabs). Lanes 2, 3, 5, 6, 7, 8, 9, 10, 12 and 13 correspond respectively to XbaI pro¢les: X1a, X1, X2, X2a, X2c, X2b, X2d, X2e, X3a and X3. Lanes 15, 16, 17, 19, 21, 22, 23 and 24 correspond respectively to SpeI pro¢les : S2, S3, S3a, S3b, S1a, S1b, S1 and S1c.
The pro¢le S3b is distinguished from S3 by the vanishing of two restriction fragments (220 and 225 kbp) and apparition of two new fragments (150 and 290 kbp) (Fig. 1, lane 19). In the pro¢le X3a, two extra bands of 340 and 500 kbp were observed (Fig. 1, lane 12). 3.2. Phage typing Phage typing was only realized on all variant isolates in order to ensure that they derived from the original vaccine or parental strains. The isolate 1607 and the nine variant isolates of M3985 had the same phagetype, DT191, identical to those of M3761 and M3985 (Table 1). Both Zoosaloral variants (1244 and 1417), the vaccine strains (Zoosaloral and vacT) and the parental strain M415 had the same phagetype DT009 (Table 1). 4. Discussion Molecular typing methods provide useful tools for an exact and reliable di¡erentiation of Salmonella vaccine strains and are helpful to check the genetic stability of live attenuated vaccines used to prevent colonization of
chickens. In the present study, two macrorestriction techniques were used to characterize three S. typhimurium vaccine strains, the parental strains and the collected isolates obtained after their passage through chickens. Genotypic analysis, by the resolution of restriction fragments of genomic DNA upon PFGE, showed that the vaccine strain M3985 is the most genetically unstable. Indeed, nine variants on 32 collected isolates were detected using SpeI or/and XbaI macrorestrictions. Di¡erences between restriction patterns were observed and implied that mutations highly occurred in vaccine strain M3985 . It is noticeable that this strain is a genetically modi¢ed organism resulting from two directed deletions (vcya vcrp) [18]. Therefore, this could a¡ect its genetic stability. However, one variant out of eight isolates collected from the wild parental strain M3761 was also observed. This phenomenon showed that the wild strain may also present some genetic instability, and might induce the instability of the derived attenuated vaccine strain M3985 . The impact of the directed deletions could also be signi¢cant. A certain genetic instability was noticed in the vaccine strain Zoosaloral: two variants out of 51 collected isolates. This result di¡ers from those obtained by Schwarz and Liebisch [19]. Using XbaI- and SpeI-macrorestriction, these authors did not observe
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Table 1 Isolates from the ¢ve Salmonella strains used for the study of genetic stability Source
Isolate
Assay
M415 845, 853, 861 871 927 1143
1 1 1 1
668, 676, 686, 695, 704, 714 622 891, 893 895, 897 899 901 1359, 1364, 1369, 1374, 1381, 1436 1438
1 2 2 2 2 2 2 2
1255, 1273, 1291 1345 1302, 1312, 1318, 1328, 1334, 1343, 1346 616, 630 883, 885, 903, 905 887, 907 889, 909 911 1218, 1220, 1228, 1230 1222, 1224, 1232, 1234 1226, 1236 1244 1246 1384, 1390 1348, 1350, 1352, 1355, 1421, 1425, 1429, 1433, 1387, 1393, 1439, 1443, 1444, 1446 1417
1 1 1 2 2 2 2 2 2 2 2 2 2 2 2
1615, 1625, 1633, 1643 1607 1723, 1731, 1739
1 1 1
1933, 1962 1946, 1954, 1968 1938, 1961 1976, 1980, 1984, 1988 638 913, 914 915 916 917 918, 919 921 1238, 1240 1241 1249 1400, 1407, 1450, 1456, 1469 1413 1463 1461
1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2
vacT
Zoosaloral
M3761
M3985
2
Sample
control caeca liver caeca caeca control caeca swab ground wall ¢lter swab caeca liver control caeca jejunum caeca swab ground wall ¢lter swab ground wall ¢lter swab swab jejunum caeca caeca control caeca caeca caeca control jejunum caeca liver liver swab ground wall wall ¢lter swab swab ground ¢lter swab caeca caeca caeca jejunum
Day of sampling
D0+72 h D0+72 h D0+6 d D0+10 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+6 w D0+6 w D0+6 d D0+10 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+5 w D0+5 w D0+5 w D0+5 w D0+5 w D0+6 w D0+6 w D0+6 w D0+6 d D0+6 d D0+10 d D0+6 d D0+6 d D0+6 d D0+10 d D0+2 w D0+4 w D0+4 w D0+4 w D0+4 w D0+4 w D0+4 w D0+5 w D0+5 w D0+5 w D0+6 w D0+6 w D0+6 w D0+6 w
Time of sampling is given as hours (h), days (d) or weeks (w) from the day of inoculation (D0) nd: not determined
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Pattern
Phage type
SpeI
XbaI
S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 S3a S3 S3 S3
X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3 X3
S3b S1 nd nd nd S1 nd nd nd nd S1a S1 S1b S1b S1b S1 S1b S1 S1 S1b S1 S1b S1c S1c
X3a X1 X1 X1a X1 X2 X2 X2 X2 X2 X2 X2 X2a X2b X2b X2 X2c X2 X2 X2b X2 X2d X2e X2e
DT009
DT009
DT009
DT009
DT009 DT191 DT191 DT191
DT191 DT191 DT191 DT191 DT191
DT191 DT191 DT191 DT191
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modi¢ed pro¢les in seven isolates of Zoosaloral collected over a 22-month period. They concluded that the Zoosaloral strain was genetically stable. It is quite interesting to note that the use of XbaI enzyme was helpful for us to detect a variant isolate of Zoosaloral. However, for Frech et al. [20], SpeI enzyme was of more interest than XbaI or BlnI to distinguish Zoosaloral strain from ¢eld DT009 strains of S. typhimurium. VacT is the only vaccine strain studied for which we did not detect variant isolates. A di¡erence between patterns of an original strain and its variants corresponds to a change in genome, so to a mutation. These variations concern only few DNA fragments, and the general pro¢le suits the original pattern: there is evidence for genomic relationship between the variants and the original strain. Moreover, all the tested isolates present the classical biochemical properties of their original vaccine strain (classical criteria of the Salmonella genus for M3761 , M415, vacT and Zoosaloral, but H2 S2 negative for M3985 ). Phage typing con¢rmed that variant isolates came from the inoculated strain. It would be then interesting to complete those results with the use of other enzymes or even other molecular tools that seem helpful to detect genomic variations among S. typhimurium strains, as IS200 typing [21]. We noticed that patterns of variant isolates were never similar to the wild strain ones (when distinguishable), so no reverse mutations were observed. Nevertheless, the origin of the mutations is unknown. It could be punctual mutations, or deletions, or even recombination with other bacterial species from the gut. It could be also mutations independent of passage on animals but connected to the long bacteriological method used for the isolation of Salmonella (in four steps, ISO 6579 :1993). The three vaccine strains are now marketed. M3985 vaccine strain is commercially available in the United States as Foodsafe 10 (Megan, USA). The other two vaccine strains are commercially available in Germany: vacT by TAD Pharmazeutisches Werk GmbH (Cuxhaven, Germany) and Zoosaloral by Impfsto¡werk Dessau-Tornau GmbH (RoMlau, Germany). Curtiss and co-workers have well studied the e¤ciency of M3985 vaccine strain [13,22]. They proved the e¤ciency of this vaccine strain to prevent colonization of the gut and to protect chicken after challenge with wild strains. On the contrary, the two other vaccine strains, vacT and Zoosaloral, appeared to be poorly inhibitory against infection with wild strains, even if they were shown to be immunogenic [23]. We were able to detect variations in the genetic stability of those vaccine strains of S. typhimurium, but we were unable to link them to a potential impact on safety or even e¤ciency. What are the real properties of these variant isolates with regards to safety, stability and e¤ciency? Would they not be better candidates for use as vaccines? We have also to keep in mind that the use of such live vaccine strains in an animal production process might
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generate hygiene and security problems for products intended to human alimentation.
References [1] Haeghebaert, S., Delarocque Astagneau, E. and Vaillant, V. (1999) Les toxi-Infections Alimentaires Collectives en France en 1997. Annual Epidemiological Report. Infectious Diseases Epidemiology in France in 1997. Re¨seau National de Sante¨ Publique, Saint-Maurice, France. [2] Bailey, J.S. (1993) Control of Salmonella and Campylobacter in poultry production, a summary of work at Russell Research Centre. Poult. Sci. 72, 1169^1173. [3] Bale, J., Bennet, P.M., Beringer, J.E. and Hinton, M.H. (1993) The survival of bacteria exposed to desiccation on surfaces associated with farm buildings. J. Appl. Bacteriol. 75, 519^528. [4] Davies, R.H. and Wray, C. (1996) Persistence of Salmonella enteritidis in poultry units and poultry food. Br. Poult. Sci. 37, 589^ 596. [5] Lahellec, C. and Colin, P. (1985) Relationship between serotypes of Salmonella from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult. Sci. 26, 179^186. [6] Cox, N.A., Bailey, J.S. and Berrang, M.E. (1996) Alternative routes for Salmonella intestinal tract colonization of chicks. J. Appl. Poult. Res. 5, 282^288. [7] Henzler, D.J. and Opitz, H.M. (1992) The role of mice in the epizootiology of Salmonella enteritidis infection in chicken layer farms. Avian Dis. 36, 625^631. [8] Wall, R.G., Davies, S., Threfall, E.J., Ward, L.R. and Ewbank, A.J. (1995) Chronic carriage of multidrug resistant Salmonella typhimurium in a cat. J. Small Anim. Pract. 36, 279^281. [9] Anonymous (1998) Arreªte¨ du 26 octobre 1998 relatif a© la lutte contre les infections a© Salmonella enteritidis ou Salmonella typhimurium dans les troupeaux de reproduction de l'espe©ce Gallus gallus en ¢lie©re chair. JORF du 8.9.98. [10] Anonymous (1998) Arreªte¨ du 26 octobre 1998 relatif a© la lutte contre les infections a© Salmonella enteritidis ou Salmonella typhimurium dans les troupeaux de l'espe©ce Gallus gallus en ¢lie©re ponte d'Eufs de consommation. JORF du 8.9.98. [11] Barrow, P.A., Lovell, M.A. and Berchieri Jr., A. (1991) The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella enteritidis phage type 4. Avian Path. 20, 681^692. [12] Cooper, G.L., Vanables, L.M., Woodward, M.J. and Hormaeche, C.E. (1994) Vaccination of chickens with strain CVL30, a genetically de¢ned Salmonella enteritidis aroA live oral vaccine candidate. Infect. Immun. 62, 4747^4754. [13] Hassan, J.O. and Curtiss III, R. (1994) Development and evalution of an experimental vaccination program using a live avirulent Salmonella typhimurium strain to protect immunized chickens against challenge with homologous and heterologous Salmonella serotypes. Infect. Immun. 62, 5519^5527. [14] Nassar, T.J., Al-Nakhli, H.M. and Al-Ogaily, Z.H. (1994) Use of live and inactivated Salmonella enteritidis phage type 4 vaccines to immunise laying hens against experimental infection. Rev. Sci Tech. O¡. Int. Epizoot. 13, 855^867. [15] Suphabphant, W., York, M.D. and Pomeroy, B.S. (1982) Use of two vaccines (live G30D or killed RW16) in the prevention of Salmonella typhimurium infections in chickens. Avian Dis. 27, 602^615. [16] Curtiss III, R., Kelly, S.M. and Hassan, J.O. (1993) Live oral avirulent Salmonella vaccines. Vet. Microbiol. 37, 397^405. [17] Anderson, E.S., Ward, L.R. and DeSaxe, J.D.H. (1977) Bacteriophage-typing designations of Salmonella typhimurium. J. Hyg. 78, 297^300. [18] Curtiss III, R. and Kelly, S.M. (1987) Salmonella typhimurium dele-
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tion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect. Immun. 55, 3035^ 3043. [19] Schwarz, S. and Liebisch, B. (1994) Pulsed-¢eld gel electrophoretic identi¢cation of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H. Lett. Appl. Microbiol. 19, 469^472. [20] Frech, G., Weide-Botjes, M., Nussbeck, E., Rabsch, W. and Schwarz, S. (1998) Molecular characterization of Salmonella enterica subsp. enterica serovar Typhimurium DT009 isolates di¡erentiation of the live vaccine strain Zoosaloral from ¢eld isolates. FEMS Microbiol. Lett. 167, 263^269. [21] Schwarz, S. and Liebisch, B. (1994) Use of ribotyping, IS200 typing
and plasmid analysis for the identi¢cation of Salmonella enterica subsp. enterica Serovar Typhimurium vaccine strain Zoosaloral H and its di¡erentiation from wild type strains of the same serovar. Zent. bl. Bakt. 281, 442^450. [22] Curtiss III, R. and Hassan, J.O. (1996) Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet. Immunol. Immunopathol. 54, 365^372. [23] Methner, U., Barrow, P.A., Martin, G. and Meyer, H. (1997) Comparative study of the protective e¡ect against Salmonella colonisation in newly hatched SPF chickens using live, attenuated Salmonella vaccine strains, wild-type Salmonella strains or a competitive exclusion product. Int. J. Food Microbiol. 35, 223^230.
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