Comparative studies on Salmonella isolations from feeds in ten laboratories*

Bull. Org. molnd. Sant) 1974, 50, 421-426Bull. Wld Hith Org. Comparative studies on Salmonella isolations from feeds in ten laboratories* W. EDEL & E. H. KAMPELMACHER On behalf of the members of a Working Group 1 Pig feed samples, artificially and naturally contaminated with S. typhimurium, were distributed to ten laboratories. The isolation rates obtained by using a standardized pre- enrichment method and the laboratories' " own " methods were compared. In nearly all laboratories the standardized pre-enrichment method gave better results. Extending the duration of incubation of the enrichment medium to 48 h followed by a second subculture improved the isolation rates. In general, a pre-enrichment method should be used for the isolation of salmonellae, especially when they are injured or small numbers of them are expected in the material to be investigated. In a previous paper (3) the isolation of salmonellae from artificially and naturally contaminated samples of minced meat, by means of both a standardized pre- enrichment method and a standardized direct enrich- ment method, was described. Those experiments showed that, especially when dealing with " suble- thally injured" salmonellae, better results were ob- tained in all laboratories by the pre-enrichment method, which yielded almost 100% recovery from artificially contaminated samples. However, by the direct enrichment method there were marked differ- ences in the results obtained from different laborato- ries. Although these differences were less marked in some laboratories, it was assumed that, in general, pre-enrichment favoured the reproducibility of the isolation rates of salmonellae, and it was recom- mended that a standardized method should always include a pre-enrichment step. In previous investigations (2, 3), only one substrate (minced meat) was examined and only one Salmonella serotype (S. utrecht) was used to contaminate sam- ples artificially. It was therefore of interest to carry out further comparative studies on another substrate * This investigation was initiated by the Common Market Scientific Veterinary Group on Salmonellosis and received financial support from the World Health Organization. Reprints may be obtained from the Laboratory for Zoonoses and Food Microbiology, National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands. 1 The names and addresses of the members of the Working Group are listed in Annex 1. with a different Salmonella type. As it is known that contaminated animal feeds play an important role in maintaining the cycles of Salmonella transmission in both animals and man, it was decided to extend the previous experiments by the use of feeds both natu- rally and artificially contaminated with S. typhimu- rium, which is the serotype most frequently isolated in most countries. The standardized pre-enrichment method was again compared with other methods of the laboratory's own choice. MATERIALS AND METHODS Animal feed samples, either artificially or naturally contaminated, were used for the experiments, which included 10 weekly investigations of 10 samples, each in duplicate, of artificially contaminated pig feeds, followed by 10 weekly investigations of another 10 samples, in duplicate, of naturally contaminated feeds. A 10 weeks' supply of artificially contaminated samples was dispatched to all the participating lab- oratories on the same day, a few weeks before the first investigation. The naturally contaminated samples were sent on two separate days, half the number of samples being sent each time and arriving a few weeks before they were needed for the experiments. The parcels containing the samples were sent directly to the different laboratories by normal air- freight and they did not have to be collected at the airport or railway station as was necessary in previous 3207 - 421- 422 W. EDEL & E. H. KAMPELMACHER investigations (1-3). After arrival at the laboratory the samples were stored in the dark at room tempera- ture until the day of investigation. The participating laboratories were the same as those mentioned in the previous publications with the exception of laboratory J. Laboratories A1, A2, and A3 were departments of the laboratory that organized the trial; the samples were prepared, packed, and dispatched by laboratory A3. Artificially contaminated samples The method used to contaminate the samples artificially has been described previously (3). S. typhi- murium, phage type I 50, was chosen for the present experiments. The milk powder containing the " suble- thally injured " Salmonella cells was initially diluted by the addition of normal spray-dried milk. In order to obtain three different levels of contamination the milk powder was thoroughly mixed with different quantities of normal pig feed purchased from a feed factory. These contained a high, moderate, or small number of Salmonella, and each mixture was dis- pensed into samples of about 25 g containing approxi- mately 200, 100, and 10 cells, respectively. The number of Salmonella cells was determined by the MPN (most probable number) method. In order to avoid cross contamination, all samples were sealed in two plastic bags, the inner bag bearing the number of the sample. In each experiment 10 samples were examined by the standardized pre-enrichment method and 10 samples by the laboratory's " own " method. The number of artificially contaminated samples varied from one experiment to another and was known only to laboratory A3, where the samples were prepared. However, in laboratory A3, the preparation of the samples was carried out by different personnel from those who subsequently performed the isolation experiments. Altogether 72 samples were artificially contami- nated for testing by both methods; these included 22, 27, and 23 containing high, moderate, and small numbers of Salmonella, respectively. Naturally contaminated feed samples Again, normal feed mixture for pigs was purchased from a feed factory. In order to obtain a sufficient number of contaminated samples of 25 g, the feeds were mixed with an aliquot of animal byproducts from a rendering plant, known to be heavily contami- nated with Salmonella. After thorough mixing the samples were weighed in quantities of approximately 25 g and put into two plastic bags, which were sealed separately. Methods of bacteriological examination All laboratories used the same standardized me- thod (pre-enrichment followed by enrichment), but additional selective plates of the laboratory's own choice could also be used. Each laboratory used an alternative " own " method, in addition to the stan- dardized pre-enrichment method. Standardized pre-enrichment method. Pre-enrich- ment of the samples in a nonselective medium was followed by enrichment in a selective medium. The entire procedure has already been described in de- tail (3). In addition to the selective plate, brilliant green phenol red lactose agar (BGA) (Oxoid CM 329), the media chosen by the laboratories were as follows: A1, A2 and A3: BGA (home-made); B: deoxycholate citrate agar, bismuth sulfite agar and BGA (Oxoid CM 263); D: Gassner plate and Pril-mannitol agar; G: BGA (Difco B285); H: two-sugar-novobiocin agar; I: BGA (home-made); C, E, F and J: none. Laboratories'" own " method. The " own " methods used by the various laboratories are summarized in Table la. RESULTS Experiments with artificially contaminated feeds By the standardized pre-enrichment method 98.2% (90.3-100 %) of the contaminated samples were found positive, while by the " own " method only 78.5% (26.4-100%) of the contaminated samples were de- tected. The results of these experiments are sum- marized in Fig. 1. Fewer samples were found to be positive when the Salmonella contamination level was reduced. This was especially true when the samples were investi- gated by the laboratories " own " methods. These results are summarized in Table 2. During the experiments, it was found that there was natural contamination of the feed mixture used for the preparation of artificially contaminated samples. Owing to this " natural " contamination of the feeds, 23 additional samples were found to be positive for a Space did not permit the publication of Tables 1, 4 and 5. These have been deposited in the WHO library and professionally interested persons may obtain single photo- copies on request to: Chief Librarian, WHO, 1211 Geneva 27, Switzerland. ISOLATION OF SALMONELLAE 00. cn 0 CL c 0 0) an 2 U,E co0 6 z 70 60 50 *40 30 20 10 0 MA':1 A21 MA3 WWB ..Cf] D :1W F1 G H '1 M. JE 0 Al A2 A3 B C D E F G tl I J i Laboratory Fig. 1. Comparison of standardized pre-enrichment and the laboratories' " own " methods for the isolation of salmonellae from artificially contamined animal feeds. The number of samples known to be contaminated was 72. Shaded columns, standardized pre-enrichment method; stippled columns, laboratories' " own " method. Table 2. Failure rates of Salmonella detection by the standardized pre-enrichment method and the laboratories'" own " methods Standardized -Own ' method No. of No. of pre-enrichment method inoculated contaminated contaminated samples contaminated samples /sample samples not detected not detected No. % No. % high (c.200) 22 x 12 = 264 4 1.5 40 15.1 moderate (c.100) 27 x 12 = 324 3 0.9 61 18.8 small (c.10) 23 x 12 = 276 9 3.3 85 30.8 Total 72 x 12 = 864 16 1.8 186 21.5 Salmonella, of which 19 (82.6 %) were isolated by the standardized pre-enrichment method and only 4 (17.4%) by the " own " method. The results of Salmonella detection from artificially and naturally contaminated samples for the various laboratories are shown in Table 3. Moreover, owing to this " natural " contamination of the feeds, 6 samples that were artificially contami- nated with only small numbers of S. typhimurium, type I 50, did not yield this serotype. Instead other serotypes were found; these samples were regarded as positive. Besides S. typhimurium, type 1 50, other sero- 423 424 W. EDEL & E. H. KAMPELMACHER Table 3. Numbers of Salmonella isolations from artificially contaminated feed samples Laboratories Ai A2 A3 B C D E F G H I J standardized pre-enrichment method: artificially contaminated 71 72 72 72 72 72 67 65 72 70 72 71 naturally contaminated a 1 2 2 1 0 3 1 2 0 4 1 2 total 72 74 74 73 72 75 68 67 72 74 73 73 "own ' method: artificially contaminated 67 72 72 53 50 43 28 70 19 67 70 67 naturally contaminated a 0 0 2 0 0 1 0 0 0 0 1 0 total 67 72 74 53 50 44 28 70 19 67 71 67 a means: additional salmonellae from natural contamination of the artificially contaminated feeds. a, 130.r ~0a c n 0 U, a, E m (AW, 0 6 z Laboratory Fig. 2. Comparison of standardized pre-enrichment and the laboratories' - own " methods for the isolation of salmonellae from naturally contaminated animal feeds. Shaded columns, standardized pre-enrichment method; stippled columns, laboratories' " own " method. types were also isolated from 8 artificially contamin- were isolated only by means of the standardized pre- ated samples, 6 of which were inoculated with small enrichment method. If the results from all the labora- and 2 with moderate numbers of Salmonella. In these tories are considered, a total of 37 samples yielded 14 naturally contaminated samples, other serotypes serotypes other than S. typhimurium, type I 50. The ISOLATION OF SALMONELLAE 425 number of samples found positive by the various laboratories are indicated in the accompanying tabul- ation. A1 A2 A3 B C D E F G H 1 J total 4 2 4 3 0 10 1 2 2 4 2 3 37 From these 37 samples a total of 41 isolates of 18 different serotypes was obtained. The distribution of the isolates according to serotype is summarized in Table 4 a. Experiments with naturally contaminated feeds With the standardized pre-enrichment method the laboratories found from 28% to 56% of the samples to be positive, with an average of 41.4%. By their " own " methods the average was 31.5 %, the isolation rates of the different laboratories ranging from 9 to 50% of the samples. The results are summarized in Fig. 2. Detailed data concerning the distribution of the isolated serotypes are given in Table 5.aAltogether 28 different serotypes were isolated, the number from individual laboratories ranging from 4 to 17. For the standardized pre-enrichment method and the " own " method these figures varied between laboratories from 4 to 12 and from 2 to 12, respectively. Influence ofthe enrichment time and the selective plates used, on the isolation of Salmonella By both methods (standardized pre-enrichment and laboratory's " own ") and with both batches of samples (artificially and naturally contaminated), similar results were obtained with the different selec- tive media used. An average of approximately 95% were found positive after incubation for 24 h, the remaining 5% being detected at the second subculture after 48 h. Only small differences in the numbers of positive samples were observed in the laboratories in which additional selective plates were used. In the previous investigation (3), these differences were greater. DISCUSSION The results of the experiments with artificially contaminated feeds show that in all laboratories almost 100% recovery was realized by means of the standardized pre-enrichment method. The recovery a See footnote on p. 422. was, in general, lower by the laboratory's " own " method, the greatest differences being observed in 5 laboratories, B-E and G (Table 3). With regard to the artificially added Salmonella cells, fewer positive samples were found at lower levels of contamination. This was especially true when the samples were investigated by the labora- tory's " own " method. The standardized pre-enrich- ment method improved the detection rate of small numbers of salmonellae. This became even more evident by comparing the standardized pre-enrich- ment method and the " own " methods in the detec- tion of natural contamination in artificially contami- nated samples. Again, it was observed that better results were obtained by extending the period of incubation of the enrichment medium to 48 h and sub-culturing a second time. The results obtained with naturally contaminated feeds were generally better by the standardized pre- enrichment than by the laboratory's " own " me- thods. Although in some laboratories there were only small differences between both methods, the results obtained between laboratories in detecting the natu- rally contaminated samples, even by the standardized pre-enrichment method, were far greater than in the experiments with artificially contaminated samples. It is of interest to consider the reasons for the discrepan- cies in the results obtained by the different laborato- ries. There are at least two possibilities. The distribu- tion of Salmonella in the feeds may have been heterogeneous; however, this is unlikely, since in 8 laboratories the results were similar by both methods. A more probable reason is that the reading of selective plates by persons not fully skilled or interested may have given less good results. Evidence for this has been observed in the departments of laboratory A over recent years. Although the amount of work and therefore the time reserved for reading one plate are important in the detection of positive samples, the human factor also seems to be highly important; this was suggested previously by Kampel- macher (4) even when a standardized method was used. In general, it may be concluded that the standard- ized pre-enrichment method gave good results and could therefore be recommended as a reference method. In addition, a pre-enrichment method should be used for the isolation of salmonellae, especially when injured salmonellae and/or small numbers of Salmonella are expected in the material to be investigated. 426 W. EDEL & E. H. KAMPELMACHER ReSUMI ETUDES COMPARATIVES SUR LES ISOLEMENTS DE SALMONELLA A PARTIR D'ALIMENTS DANS DIX LABORATOIRES On a distribue a 10 laboratoires des echantillons d'ali- ments pour porcs, artificiellement (10, 100 ou 200 cel- lules environ par echantillon) ou naturellement conta- mines par Salmonella typhimurium, et compare les taux d'isolement obtenus en utilisant une methode de preen- richissement normalisee ou d'autres methodes choisies par les laboratoires. Avec la methode normalisee, les differents laboratoires ont trouve 90,3 a 100% d'echantillons artificiellement contamines positifs; avec leurs methodes propres, ils n'en ont decouvert que 26,4 'a 100%. Les taux d'echecs de la methode normalisee ont ete de 1,5 %, 0,9% et 3,3% respec- tivement pour les specimens fortement, moderement ou faiblement contamines. Avec les autres methodes, les chif- fres correspondants ont ete de 15,1%, 18,8% et 30,8%. Les echantillons naturellement contamines ont donne 28 a 56% de resultats positifs par la methode normalisee, avec une moyenne de 41,4%. Par les autres methodes, on a obtenu une moyenne de 31,5% d'isolements posi- tifs, le taux variant de 9 a 50% selon les laboratoires. Les taux d'isolement ont ete ameliores lorsqu'on a porte la duree de la periode d'incubation en milieu d'enrichissement a 48 heures et procede a une seconde sous-culture. En general, il convient d'utiliser une methode de preenrichissement pour l'isolement des Salmonella, en particulier lorsqu'on pressent que le materiel a examiner renferme des organismes leses ou en petit nombre. REFERENCES 1. EDEL, W. & KAMPELMACHER, E. H. Bull. Wld Hlth Org., 39: 487 (1968). 2. EDEL, W. & KAMPELMACHER, E. H. Bull. Wld Hlth Org., 41: 297 (1969). 3. EDEL, W. & KAMPELMACHER, E. H. Bull. Wld Hlth Org., 48: 167 (1973). 4. KAMPELMACHER, E. H. Zbl. Bakt., L Abt. Orig., 204: 100 (1967). Annex I MEMBERS OF THE WORKING GROUP M. Baroutchieva, Bacteriologist, Kimron Veterinary Institute, Bet Dagan, Israel E. Bulling, Director and Professor, Federal Institute of Public Health, Robert von Ostertag Institute for Veterinary Medicine, Berlin W. Edel, Bacteriologist, Laboratory for Zoonoses and Food Microbiology, National Institute of Public Health, Bilthoven, The Netherlands A. C. Ghosh, Food Hygiene Laboratory, Central Public Health Laboratory, London, England G. Gottal, Director, National Veterinary Medical Labo- ratory, Luxembourg B. C. Hobbs, Head, Food Hygiene Laboratory, Central Public Health Laboratory, London, England E. H. Kampelmacher, Head, Laboratory for Zoonoses and Food Microbiology, National Institute of Public Health, Bilthoven, The Netherlands; Extraordinary Professor, Agricultural University, Wageningen, The Netherlands G. Lott, Head, Meat Hygiene Laboratory, Veterinary Bacteriological Institute, University of Zurich, Switzer- land A. Linton, Senior Lecturer in Veterinary Bacteriology, Medical School, University of Bristol, England J. Pantaleon, Scientific Director, Central Laboratory of Veterinary Research, Alfort, France M. van Schothorst, Bacteriologist, Laboratory for Zoo- noses and Food Microbiology, National Institute of Public Health, Bilthoven, The Netherlands G. Tiecco, Institute of Public Health, Rome, Italy J. Thomas, Head, Bacteriological Department, National Institute of Veterinary Research, Brussels, Belgium