Comparison of Selective Enrichment Broths for Detection of Salmonella in Animal Feed

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1 Jpn. J. Food Microbiol., 28(3), , , Comparison of Selective Enrichment Broths for Detection of Salmonella in Animal Feed Tetsuo C=>=6G6 1,, Satomi T6C6@6 1 and Toshiharu Y6<> 2 ( 1 Food and Agricultural Materials Inspection Center, Kobe Regional Center, Minatojima-minami-machi, Chuo-ku, Kobe-shi, Hyogo ; Corresponding author) ( 2 Food and Agricultural Materials Inspection Center, Shintoshin, Chuo-ku, Saitama-shi, Saitama ) Four selective enrichment broths were compared to improve the O$cial Method in Japan for detection of Salmonella in feed ingredients and formula feed. The current O$cial Method specifies the use of both Hajna tetrathionate broth (HTT) and selenite cystine broth (SC) for selective enrichment following pre-enrichment. Because SC contains toxic sodium selenite, Rappaport Vassiliadis enrichment broth (RV) and Rappaport Vassiliadis soya peptone broth (RVS) were evaluated as alternatives. In a comparison study of spiked broths, 100 ml of RV and RVS were inoculated with 1 ml or 10 ml of pre-enrichment culture. When inoculated with culture that included Salmonella, there were no di#erences in Salmonella growth. When inoculated with Salmonella only, the Salmonella count in SC, RV and RVS reached 10 8 cfu/ml, but only 10 7 cfu/ml in HTT. When inoculated with cultures containing both Salmonella and a member of the coliform group, the Salmonella count remained about the same while the coliform group count reached cfu/ml. The result showed that all four broths selectively increased Salmonella growth even in the presence of a coliform competitor. In a study on naturally contaminated feed, 13 out of 23 samples were Salmonella positive (56.5 ) with all four broths. A delayed secondary enrichment on the 10 negative samples did not change those results. Comparison studies on routinely inspected feed showed that 6 of 288 samples were Salmonella positive (2.1 ) using HTT, SC and RV and 12 of 404 samples were Salmonella positive (3.0 ) using HTT, SC and RVS. Further comparison of those results against HTT, SC and RV or RVS showed no significant di#erence between the broths (p 0.05). In an inter-laboratory comparison, all six laboratories found consistent Salmonella-positive and -negative results for all four broths. These results demonstrate that RV and RVS are acceptable alternatives to SC as selective enrichment broths for detection of Salmonella in feed. Key words: Salmonella, selective enrichment broth, comparison study, animal feed

2 176 Vol. 28 No ) (FAMIC) 22) /2, /1, /2, /272 1, 4, 9, 19, 21) 2001 BSE 19) 14) ) 15) 20) (HTT) (SC) SC 5) 23) SC ISO ) (RVS) FAMIC 1996 ISO 2007 SC ISO 6579 RVS SC RVS ) (RV) ISO ), AOAC O$cial Method ) RV RV RVS 17, 25) SC RV RVS (4 1 ) 2. Salmonella Abony ACM 5080 BioBall SingleShot 30 (bio- Mérieux), 30 cfu/ O ml 3cfu/ml Tween 80 (BPW) BPW Bacto-Peptone (Difco) HTT SC Selenite cystine broth (Difco), RV Rappaport Vassiliadis enrichment broth (Oxoid) RVS Rappaport Vassiliadis soya peptone broth (Oxoid) HTT SC RV RVS DHL DHL; Brilliant green agar (BG; Difco) CHROMagar Salmonella (CAS; CHROMagar) TSI TSI; SIM SIM; Lysine decarboxylase broth (LDB; Difco) ) Fig. 1 HTT SC RV RVS 100 ml, 10 ml DSE 8, 12) HTT ml HTT 9 ml

3 177 Fig. 1. Procedure for comparison tests of the selective enrichment broth to detect Salmonella in feed and for confirmation of detection precision between laboratories Extended secondary enrichment used only when selective enrichment of naturally contaminated samples was negative-not used for confirmation of detection precision. For comparison selective enrichment broth was added to the O$cial Method. BPW: bu#ered peptone water with 0.6 tween 80, HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RV: Rappaport Vassiliadis enrichment broth, RVS: Rappaport Vassiliadis soya peptone broth, DHL: desoxycholate hydrogen sulfide lactose agar, BG: brilliant green agar, CAS: chromagar Salmonella agar, TSI: triple sugar iron agar, SIM: sulfide indole motility agar, LDB: lysine decarboxylase broth 2) a) 10 g 100 ml 10 Heart infusion agar (Difco) 12) Desoxycholate agar (Difco) 13) b) 1ml, 25 g 1ml 25 g BPW 250 ml 10 ml HTT, SC, RV RVS 100 ml 10) ml CAS 3) RV RVS RV RVS 1/100 1/10 1ml 10 ml RV RVS 100 ml 1ml 10 ml RV RVS 100 ml 4) RV RVS 5) SC SC RV RVS HTT SC RV HTT SC RV RVS HTT SC RVS FAMIC 6 SC

4 178 Vol. 28 No ) FAMIC 6 25 g 1ml 25 g 3 RV RVS DHL, BG CAS TSI, SIM LDB FAMIC 6 O4 1. RV RVS 1ml 10 ml RV RVS Fig cfu/ml, cfu/ml, cfu/ml cfu/ml cfu/g, cfu/g 2. Fig cfu/ml, SC, RV RVS cfu/ml HTT cfu/ml cfu/ml cfu/ml, cfu/ml cfu/ml cfu/ml, cfu/ml cfu/ml, cfu/ml, cfu/ml cfu/ml 3. HTT, SC, RV RVS Table /20 1/ DSE 4. SC RV Table 2 RV /158 1/ (HTT: Fig. 2. Comparison of total viable count, coliform group count and Salmonella count in the culture from RV or RVS Salmonella Abony ACM 5080 (3 cfu) or layer feed (25 g) spiked with Salmonella Abony ACM 5080 (3 cfu), was pre-enriched by BPW. And 1 ml or 10 ml of the culture from BPW was inoculated into 100 ml of RV or RVS. In layer feed total viable count on HIA and DCA were and cfu/g, respectively. a: Total viable count on HIA, b: Total viable count of coliform group including Salmonella on CAS, c: Total viable count of Salmonella (purple colony) on CAS BPW: bu#ered peptone water with 0.6 tween 80, RV: Rappaport Vassiliadis enrichment broth, RVS: Rappaport Vassiliadis soya peptone broth, HIA: heart infusion agar plate, DCA: desoxycholate agar plate, CAS: chromagar Salmonella agar plate

5 179 Fig. 3. Comparison of total viable count, coliform group count and Salmonella count in the culture from enrichment broth Salmonella Abony ACM 5080 (3 cfu), layer feed (25 g) or layer feed (25 g) spiked with Salmonella Abony ACM 5080 (3 cfu), was pre-enriched by BPW. And 10 ml of the culture from BPW was inoculated into 100 ml of HTT, SC, RV or RVS. In layer feed total viable count on HIA and DCA were and cfu/g, respectively. a: Total viable count on HIA, b: Total viable count of coliform group including Salmonella on CAS, c: Total viable count of Salmonella (purple colony) on CAS BPW: bu#ered peptone water with 0.6 tween 80, HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RV : Rappaport Vassiliadis enrichment broth, RVS: Rappaport Vassiliadis soya peptone broth, HIA: heart infusion agar plate, DCA: desoxycholate agar plate, CAS: chromagar Salmonella agar plate 1.7, SC: 1.7, RV: 1.7, HTT&SC: 2.1, HTT&RV: 1.7 ) 6 1 SC HTT RV 1 HTT RV SC HTT,SC RV (p 0.05) SC 1 RVS Table 3 RVS /225 2/ (HTT: 3.0, SC: 2.7, RVS: 3.0, HTT&SC: 3.0, HTT&RVS: 3.0 ) 12 1 HTT RVS SC HTT, SC RVS (p 0.05) SC 5. FAMIC 6 FAMIC 6 Table 4 DHL, BG CAS 3 DHL BG 1 BG DHL CAS 7) 24) RV RVS 1/100 2, 3, 6) RV RVS 1/100, 1/10

6 180 Vol. 28 No Table 1. Detection of Salmonella from naturally contaminated feed using selective enrichment media, HTT, SC, RV or RVS Kind of feed Number of samples Result in selective enrichment broth Examined Positive Negative HTT SC RV RVS Result of DSE method Feed ingredient Feather meal 2 Fish meal Meat and bone meal (derived from pork and poultry) Poultry by-product meal 2 Soybean meal Formula feed For poultry For swine 1 For cattle Total Positive rate ( ) : Positive, : Negative HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RV: Rappaport Vassiliadis enrichment broth, RVS: Rappaport Vassiliadis soya peptone broth, DSE: delayed secondary enrichment 1/10 RV RVS 1/10 RV 24) Table 1 3 RV RVS RV RVS 10 8 cfu/ml 10 6 cfu/ml SC, RV RVS 10 8 cfu/ml HTT 10 7 cfu/ml HTT 29 HTT HTT, SC, RV RVS 10 8 cfu/ml 18) DHL, BG CAS 10 3 cfu/ ml DSE 2 cfu/100 g 4)

7 181 Table 2. Kind of feed Detection of Salmonella from routinely inspected feed using selective enrichment media, HTT, SC or RV Number of samples Result of positive sample in selective enrichment broth Examined Positive HTT SC RV HTT&SC b HTT&RV b Feed ingredient Animal protein feed Animal protein and soybean meal mixed feed 3 0 Feather meal 16 0 Fish extraction processed by enzyme 1 0 Fish meal Fish scales extraction 1 0 Fishery animal fermented 3 0 Meat and bone meal (derived from pork) 2 0 Meat and bone meal (derived from pork and poultry) 16 0 Shrimp meal 1 0 Squid meal 1 0 Poultry by-product meal 24 1 Oil seed meal 15 1 Corn germ meal 1 0 Linseed meal 1 0 Rapeseed meal 1 0 Sesame meal 2 0 Soybean meal 9 0 Soybean meal (dehulled) fermented 1 1 Bran and food processing by-product 3 0 Corn gluten feed 1 0 Distiller s dried grains with soluble 1 0 Rice bran 1 0 Formula feed For poultry 52 1 a For swine 28 0 For cattle 50 0 Total Positive rate ( ) a For layer, b Judged by two broths : Positive, : Negative HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RV: Rappaport Vassiliadis enrichment broth RV RVS HTT SC RV RVS FAMIC 6 SC RV RVS SC RV ) RV RVS SC 1) RV RVS 1/100, 1/10 2) 10 8 cfu/ml 10 6 cfu/ml

8 182 Vol. 28 No Table 3. Kind of feed Detection of Salmonella from routinely inspected feed using selective enrichment media, HTT, SC or RVS Number of samples Result of positive sample in selective enrichment broth Examined Positive HTT SC RVS HTT&SC c HTT&RVS c Feed ingredient Animal protein feed Animal protein and soybean meal mixed feed 3 1 Crab shell meal 3 0 Feather meal 16 2 Fish meal Fish scales extraction 1 0 Fishery animal fermented 1 0 Meat and bone meal (derived from pork) 2 0 Meat and bone meal (derived from pork and poultry) 21 4 Squid meal 2 0 Poultry by-product meal 26 0 Oil seed meal 21 1 Copra meal 1 0 Corn gluten meal 1 0 Molasses absorbed feed 2 0 Rapeseed meal 5 0 Sesame meal 3 0 Soybean meal 8 1 Soybean protein concentrate 1 0 Bran and food processing by-product 12 0 Brewer s grain 2 0 Corn gluten feed 1 0 Distiller s dried grains with soluble 2 0 Rice bran 4 0 Soy sauce cake 1 0 Wheat bran 2 0 Others 2 0 Fish meal and maize mixed feed 1 0 Sweet potato 1 0 Formula feed For poultry 70 1 a For swine 45 1 b For cattle 64 0 Total Positive rate ( ) a For quail, b For finishing pig, c Judged by two broths : Positive, : Negative HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RVS: Rappaport Vassiliadis soya peptone broth 3) SC,RV RVS 10 8 cfu/ml HTT 10 7 cfu/ml cfu/ml 4) (56.5 ) HTT, SC, RV RVS 10 DSE 5) RV RVS RV 2.1 6/288 RVS /404 (p 0.05).

9 1) Unspiked Result of selective isolation Result of biochemical characteristics test a Last judgment result Table 4. Confirmation of Salmonella detection precision between laboratories Repetition frequency Laboratory HTT SC RV RVS HTT SC RV RVS HTT SC RV RVS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS A B C D E F A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N B N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N C N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N D N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N E N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N F N N N N N N N N N N N N N N N N N N N N N N A B C D E F 183

10 2) Spiked with Salmonella Abony ACM 5080 (3 cfu/25 g sample) Table 4. Continued Repetition frequency Laboratory HTT SC RV RVS HTT SC RV RVS HTT SC RV RVS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS DHL BG CAS Result of selective isolation A B C D E F Result of biochemical characteristics test a A B C D E N F Last judgment result b A B C D E F a A result of biochemical characteristics test in TSI, SIM and LDB, b A result of serological test with somatic O4 antiserum (excluding negative case) : Judged to be pseudo-positive, : Judged to be positive, : Judged to be negative, N: Not tested HTT: Hajna tetrathionate broth, SC: selenite cystine broth, RV: Rappaport Vassiliadis enrichment broth, RVS: Rappaport Vassiliadis soya peptone broth DHL: desoxycholate hydrogen sulfide lactose agar, BG: brilliant green agar, CAS: chromagar Salmonella agar TSI: triple sugar iron agar, SIM: sulfide indole motility agar, LDB: lysine decarboxylase broth 184 Vol. 28 No

11 185 6) FAMIC 6 FAMIC 6 1) 19 33, (2008). 2) Andrews, W. H. and Hammack, T. S.: Bacteriological Analytical Manual. Chapter 5 Salmonella. U.S. Food and Drug Administration (2006). 3) AOAC O$cial Method : Salmonella in Raw, Highly Contaminated Foods and Poultry Feed. AOAC OFFICIAL METHOD OF ANALYSIS, Chapter 17, (2005). 4) TM 25, (2008). 5) (1950). 6) International Organization for Standardization, EN ISO Microbiology of food and animal feeding stu#s. Horizontal method for the detection of Salmonella spp. ISO, Geneva, Switzerland (2002). 7) 55, (1981). 8) 37, (2001). 9) 14, (1989). 10) p , (2004). 11) 26, (2009). 12) 27, (2010). 13) p , (2005). 14) (1998). 15) (2008). 16) (2010). 17) Peterz, M., Wiberg, C. and Norberg, P.: The e#ect of incubation temperature and magnesium chloride concentration on growth of Salmonella in home-made and in commercially available dehydrated Rappaport Vassiliadis broths. J. Appl. Bacteriol., 66, (1989). 18) p , (1978). 19) 39, (2003). 20) 18 p. 6 10, (1980). 21) 20 34, (2009). 22) (1953). 23) (1999). 24) von Schothorst, M. and Renaud, A.: Dynamics of Salmonella isolation with modified Rappaport s medium (R10). J. Appl. Bacteriol., 54, (1983). 25) von Schothorst, M., Renaud, A. and von Beekb, C.: Salmonella isolation using RVS broth and MLCB agar purchase. Food Microbiology, 4, (1987).

Microbiological Methods V-A- 1 SALMONELLA SPECIES PRESUMPTIVE AND CONFIRMATION TESTS

Microbiological Methods V-A- 1 PRESUMPTIVE AND CONFIRMATION TESTS PRINCIPLE SCOPE Enrichment and selective procedures are used to provide a reasonably sensitive, definitive and versatile means of qualitatively