lnternattonal Journal of Food M:crob:ologv, 12 (1991) 257-262 ~ 1991 EIsewer Science Publishers B.V. 0168-1605/91/$03.50

257

F O O D 00386

Survey of the incidence of Listeria monocytogenes and other Listeria spp. in experimentally irradiated and in matched unirradiated raw chickens Stephanie J. Lewis and Janet E.L. Corry

*

Food Sc:ence Dw:s:on, Mm:stry of Agrteulture, Ftsherles and Food, Colney Lane. Norwich. U.K. (Received II July 1990; accepted 30 October 1990)

l.astena spp. were sought in 32 chickens experimentally irradiated (2.5 kGy) and in 25 matched unirradiated chickens. Using the modified Food and Drug A d n u m s t r a t i o n method only three of the chickens were positive for L. monocytogenes. Using the more sensitive cold enrichment method 30 were found positive. The proportion of carcasses contaminated with L. monocytogenes was lower m the ~rradiated birds than in the umrradiated birds. The results also indicated that the n u m b e r s of L. monocytogenes in the irradiated birds were lower than in the unirradiated birds. T h e only other species of Ltsteria isolated was L, mnocua, which was found on 44~ of unirradiated chickens, but on none of the irradiated chickens. Key words. L:sterm monocytogenes; l.asteria mnocua; Radiation resistance; Chacken; Cold e n n c h m e n t

Introduction A recent W H O report (WHO, 1988) recommended that food irradiation, as well as other bactericidal procedures, be investigated to determine their adequacy in the destruction of Listeria monocytogenes. In order to investigate the efficacy of such treatment, the incidence of Listeria spp. was studied in raw chickens which had been irradiated to a dose of 2.5 k G y using a Cobalt-60 source. Samples were taken from experimentally irradiated and unirradiated chickens stored at 4 ° C at intervals for up to 15 days.

Materials and Methods

Irradiatmn and Storage Sixty-four raw chickens, obtained individually packed (sealed in plastic) from three different poultry processing plants were transported chilled to Isotron Ltd.

Correspondence (Present) address." S.J. Lewis, R H M Research and Engineering, Lord Rank Research Centre. Lincoln Road, H.gh Wycombe. Buckinghamshire HP2 3QR, U.K. • Present address: J. Sainsbury plc, Stamford House, Stamford Street, London SE1 9LL, U.K.

258 (Swindon, Wiltshire) where 32 were irradiated to a dose of 2.5 kGy using a Co-60 source. The birds were transported (still in their original packing) to the laboratory in separate chilled cool-boxes for irradiated and unirradiated birds and were received within 22 h of irradiation. The chickens were kept individually wrapped and were stored at 4 ° C for 1, 3, 5.7, 9, 11 and 15 days. The unirradiated chickens were treated in the same way as the irradiated birds, although not sampled after day 9 as by that time extensive spoilage had occurred. On any one day, the control and irradiated birds that were examined originated from the same poultr3' processor. Each chicken was sampled on only one occasion. Samphng A 10 g portion of neck-flap was taken aseptically and divided into two 5 g portions. Each portion was subjected to analysis for the presence of listerme using one of the methods described below in addition to direct plating. Methods o f tsolat~on and identification Two methods of isolation with enrichment, and one direct plating method were used in parallel. The enrichment methods were: (a) the slightly modified United States Food and Drug Administration Method ( F D A M ) and (b) a cold enrichment technique (CE). The methods were as described in Lewis and Corry (1991), but modified as follows: 5 g of chicken was added to 45 ml of enrichment broth. For direct plating, duplicate 0.1 ml volumes of the one in ten suspension of chicken in nutrient broth were spread on modified McBride agar (MMA, Oxoid) and incubated at 37 ° C for 24 h. Identification methods were as described by Lewis and Corry, (1991). Isolates considered to be L. monocytogenes were sent to the Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London, for confirmauon of identity and serotype.

Results The results of the microbiological examination of irradiated and 'control', unirradiated, chickens are summarised in Table I and given m detail in Table II, which also shows the serotypes of L. rnonocvtogenes detected.

TABLE 1 Occurrence of Ltsterta spp. and /., monocvtogenes m raw chickens

Irradiated cluckens Umrradlated chickens

No. of samples tested

No. (~-) of samples containing Ltsterla spp.

No (%) of samples containing L monocvtogenes

32

11 (34)

11 ~34)

25

19 (76)

14 (56)

259 As might be expected from the ubiquitous distribution of Listerta spp., small numbers of these organisms were detectable in a significant proportion (8 out of 12) of the 'control" birds after 1 day's storage at 4 ° C; at (and beyond) 5 days' storage they were detectable in all the control birds examined. With the exception of one bird which appeared to be heavily contaminated at 5 days (detectable by direct plating, see Table II, listeriae were not present in unirradiated birds in numbers sufficient to be detected by the modified F D A method until 5 days' storage had elapsed. After 9 days, microbial spoilage of the unirradiated birds was extensive and so no detailed examination was attempted on the remaining five unirradiated birds. Listeria spp. were not detected after 1 day storage in 11 out of the 12 birds which had been irradiated. In the 12th bird they were detected only after 4 weeks of cold-enrichment. Storage of the irradiated birds at 4 0 C was accompanied by some development of Ltsteria spp. However, the organism was found in only five of 23 chickens sampled after 7 days' storage. They were detected by the modified F D A method only after 15 days' storage. It is clear that under the conditions employed a 2.5 k G y dose, whilst not sufficient to kill all listeriae, causes a major reduction in their numbers and for the first 3 days of storage at 4 ° C they remain virtually undetectable. On the basis of the limited number of birds examined the level of contamination in irradiated birds after 5 - 7 days' storage is no greater than that in the control birds after 1 day.

Discussion

L. monocytogenes was found in 56% of unirradiated birds. This is similar to the incidence found by Pini and Gilbert (1988) who found 60% of raw unirradiated chickens carried L. monocytogenes. L. innocua was the only other Listeria sp. detected (in 34% of the unirradiated birds.) Listeriae were isolated by direct plating on only one occasion and with that exception it is likely that, when present, there were < 102 listeriae per g of food. The one exception to this was an unirradiated chicken from which L. monocytogenes was isolated by direct plating after the chicken had been stored for 5 days at 4 ° C. It is probable that storage at 4 ° C permitted growth and since this was an isolated observation it is likely that this one chicken originally contained an unusually high level of iisteriae. The manner in which the chickens were obtained precluded sampling the same birds before and after irradiation. However, sufficient numbers of irradiated and unirradiated chickens were sampled to demonstrate the effects of the irradiation treatment used. Thirty-four per cent of irradiated birds contained L. monocytogenes compared to 56% of unirradiated birds. Unirradiated chickens contained L monocytogenes and L. innocua, whereas /_ monocytogenes only was found in irradiated chickens, indicating that /_ monocytogenes may be more radiation resistant than L. innocua. Stegeman (1988) presented results indicating that D-values for L. monocytogenes were similar to those for Salmonella typhimurium for radiation doses of 0.2-1.06

Unirradlated chickens

Irradiated chickens

12 (9)

2 (2)

4 (2)

!

1 1

15

7 9 9 I1 I1 15

5 7

4 ( 1) 4 (2)

3 (2)

1 3

at 4 ° C

examined (No. containing Ltsterta )

12 (1) 3 (1)

Days of storage

No. of chickens

monocytogenes monocytogenes mnocua mnocua

monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes

Species

Species and serotype of isolates from chickens

TABLE I1

1/2 I/2

4b 1/2 4b 4b 1/2 4b 4b 4b 1/2 4b !/2 1/2 4b 4b

Serotype DP a

FDAM a

Method of isolation (week of enrichment) CE(0)

CE(I)

CE(2)

9

7

5 5 7

5

3 5

3

IFIflOCUO

monoc),togene$

inrlocua

irm~ua

monocytogenes monocytogenes monocytogenes innocua innocua

Jnnocua

I/2

1/2 4b 1/2

l /2 I/2

monocylogenes monocytogenes

innoc~

l/2 1/2

I/2 4b I/2

1/2 4b 4b

monocytogene$ monocytogenes

it?nocua

monocytogenes monocytogenes monocytogenex innocua mnocua innocua monocytogene$ monocytogenes monocytogene$

• DP, direct plating; FDAM-Modificd FDA method; CE, cold enrichment.

i (])

4 (2)

4 (4)

4 (3)

1 I | i I | 3

262

k G y . S i m i l a r results were r e p o r t e d by T a r j a n (1988) a n d P a t t e r s o n (1989). H o w e v e r . in all of these studies liquid c u l t u r e media, o r buffer, a n d sterile c o m m i n u t e d m e a t s were artificially c o n t a m i n a t e d with L. m o n o c y t o g e n e s whilst n a t u r a l l y c o n t a m i n a t e d chickens were used in the s t u d y d e s c r i b e d here. T h e results in the p r e s e n t s t u d y i n d i c a t e that a r a d i a t i o n d o s e o f 2.5 k G y w o u l d reduce n u m b e r s of L. m o n o c v m g e n e s b u t n o t e l i m i n a t e it c o m p l e t e l y f r o m raw chicken. I n general, listeriae were isolated f r o m u n i r r a d i a t e d chickens s o o n e r than from i r r a d i a t e d chickens. This is t a k e n to reflect the fact that the low n u m b e r s of o r g a n i s m s which survived i r r a d i a t i o n w o u l d t a k e l o n g e r to m u l t i p l y to d e t e c t a b l e n u m b e r s . It m a y also reflect the r e s u s c i t a t i o n of r a d i a t i o n - d a m a g e d o r g a n i s m s d u r i n g p r o l o n g e d s t o r a g e at 4 ° C. F u t u r e w o r k on the effect of the f o o d m a t r i x on the r a d i a t i o n resistance o f listeriae s h o u l d be u n d e r t a k e n to investigate this possibility. A s in o t h e r recent w o r k on retail food s a m p l e s (Lewis a n d C o r r y , 1991), the c o l d e n r i c h m e n t m e t h o d used here resulted in a m u c h larger n u m b e r of p o s i t i v e s a m p l e s t h a n the F D A M m e t h o d . In the p r e s e n t w o r k the c o l d e n r i c h m e n t m e t h o d s h o w e d that 30 o f the c h i c k e n s c o n t a i n e d L. m o n o c y t o g e n e s w h e r e a s o n l y three were positive using the F D A M . O n e o f these three was negative, however, b y the c o l d e n r i c h m e n t method. In concluding, the p r e s e n t i n v e s t i g a t i o n s have s h o w n that i r r a d i a t i o n to a d o s e o f 2.5 k G y (which has b e e n suggested as a t r e a t m e n t to e l i m i n a t e s a l m o n e l l a e f r o m raw chickens) has a significant effect in r e d u c i n g the i n c i d e n c e of L i s t e r i a l spp. in fresh chicken a n d d e l a y s the s u b s e q u e n t d e v e l o p m e n t of these o r g a n i s m s d u r i n g s t o r a g e at 4 ° C .

Acknowledgements W e w o u l d like to t h a n k Dr. J. M c L a u g h l i n a n d Dr. A . G . T a y l o r , D i v i s i o n of M i c r o b i o l o g i c a l R e a g e n t s a n d Q u a l i t y C o n t r o l , C e n t r a l Public H e a l t h L a b o r a t o r y , for s e r o t y p i n g the strains. References Lewts. S.J. and Corry, J.E.L. (1991) Comparison of a cold enrichment and the FDA method for tsolatmg Lasterta monocytogenes and other Lasterta spp. from ready-to-eat food on retail sale m the U.K. Int. J. Food Microbiol. 12, 281-286. Lovett, J.. Francis, D.W. and Hunt. J.M. (1987) Ltstena monocytogenes m raw milk: detection, incidence and pathogenictty. J. Food Protect. 50. 188-192. Patterson, M. (1989) Sensitivity of l.~stena raonocytogenes to ~rradmtmn on poultry meat and in phosphate buffered saline, l.,ctt. Appl. Microbiol. 8, 181-184. Ptm, P.N. and Gilbert, R.J. (1988) The occurrence in the U.K. of Lasterm species m raw chickens and soft cheeses. Int. J. Food Microbiol. 6, 317-326. Stegeman, H. (1988) Radiation resistance of l.astena monocytogenes. Poster paper presented at the Xth International Symposium on Problems of Listeriosis, 22-25 August 1988, Pecs, Hungary. Tar'Jan, V. (1988) The sensitivity of l.,ister:a monocytogenes to gamma radiation. Poster paper presented at the Xth International Symposium on Problems of Ltsteriosis, 22-25 August, Pecs, Hungary. World Health Organization (1988) Report of the Informal Working Group on Foodborne Listenosis, Geneva, 15-19 February. WHO/EHE/FOS/88.5.

Survey of the incidence of Listeria monocytogenes and other Listeria spp. in experimentally irradiated and in matched unirradiated raw chickens.

Listeria spp. were sought in 32 chickens experimentally irradiated (2.5 kGy) and in 25 matched unirradiated chickens. Using the modified Food and Drug...
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