lmernattonal Journal of Food Mtcroblology. 12 (1991) 173-180 © 1991 Elsevier Science Pubhshers B.V. 0168-1605/91/$03.50
173
FOOD 00355
The incidence of Listeria monocytogenes in slaughtered animals, in meat, and in meat products in Yugoslavia Sava Bun6i6 Umverstty of Belgrade, Veterinary Faculty, Department for Meat Hygtene and Technology, Belgrade, Yugoslavia
(Received 7 February 1990: accepted 6 August 1990)
45% of all pigs examined harboured L. monocytogenes m the tonsils, and 3% were faecal excretors. L. monocytogenes was demonstrated m 29% of swabs from the retropharyngeal nodes of cattle and in 19% of faecal samples. The tonsillar and retropharyngeal samples did not correspond to the faecal samples. L. monocytogenes was not demonstrated in the deeper parts of the muscle tissue from 12 beef carcasses all harbouring ~steria in lymph nodes. L. monocytogenes was found in 69% of minced meat (mixed pork and beef) samples. 19% of raw dry sausages and 21% of vacuum-packaged hot smoked sausages were positive for L. monocytogenes. 1_. monocytogenes was not detected in the hot smoked sausages heated to an internal temperature of 70-75°C, after the smokang process. Key words: i.asterta monocytogenes; Swine tonsils; Retropharyngeal nodes; Meat; Meat products
Introduction T h e r e are a n u m b e r of data c o n f i r m i n g the fact that Listeria m o n o c y t o g e n e s is a f o o d - b o r n e p a t h o g e n (Schlech et al., 1983, F l e m i n g et al., 1985; J a m e s et al., 1985; Sch~Snberg, 1988). Implicated foods have been milk a n d dairy products, vegetables, a n d salads. Recently, some i n f o r m a t i o n i n d i c a t i n g that meat a n d p o u l t r y p r o d u c t s c a n also be vectors for h u m a n listeriosis outbreaks, has been reported (Barnes et al., 1989; Leistner et al., 1989; G i l b e r t et al., 1989). H u m a n listeriosis has hitherto not been a notifiable disease in Yugoslavia, a n d no reliable data are available. A n i m a l listeriosis in Yugoslavia has o n l y relatively s e l d o m been reported. Between 1978 a n d 1988 only 24 cases in cattle were registered, 12 in sheep, 1 in a horse, a n d 1 in deer: n o cases of listeriosis have been registered in pigs. F r o m a meat hygiene p o i n t of view, healthy carriers of L. m o n o c y t o g e n e s a m o n g a n i m a l s are i m p o r t a n t as a possible source of c o n t a m i n a t i o n of carcasses in slaughterhouses a n d the processing e n v i r o n m e n t .
Correspondence address: S. Bun~i~. Umversity of Belgrade. Veterinary Faculty, Department for Meat Hygiene and Technology, Bul. JNA 18. 11000 Belgrade, Yugoslavta.
174
I_. monocytogenes has been isolated from the intestinal tract of cattle (Cotin et al.. 1985; Hofer, 1983; Kampelmacher and Van Noorle Jansen, 1969; Skovgaard and Morgen, 1988), sheep (Gr~SnstiSl, 1979), poultry (Larsen, 1966; Skovgaard and Morgen, 1988), and pigs (Larsen, 1966; Skovgaard and NiSrrung, 1989) in different countries. There seem, however, to be no reported data in literature on the prevalence of L. monocytogenes in healthy animals and in meat and meat products in Yugoslavia. The present investigations were initiated to investigate the prevalence of L. monocvtogenes in slaughtered pigs, in young cattle, and in related meat and meat products.
Materials and Methods
Tonsds swabs from pigs Swabs were taken on the slaughter line of a modern slaughterhouse, both from the surface and from fresh cuts of the tonsils from 103 pigs originating from 36 different private farms. The farms represented two groups: in Group I the pigs were fed predominantly on silage, in Group II they were fed dry feed only. The swabs were immediately placed in 10 ml of Listeria Enrichment Broth I (LEB I), followed by subsequent subcultivation in LEB II and isolation and confirmation according to Skovgaard and Morgen (1988) and as described below. Lymph node swabs from cattle Swabs of both the surface and of fresh cuts of the internal retropharyngeal nodes (IRN) were taken from 52 young cattle (bullocks and heifers at the age of 18 months) on the slaughter line of a slaughterhouse. The swabs were placed in 10 ml of LEB I and cultivated as described below. Faecal samples from pigs and cattle Approximately 25 g of faeces were collected on the slaughter line from the lower part of the intestinal tract of 97 pigs originating from two different state-owned farms, A and B. At farm A only dry feed was used, and at farm B silage was also used. Faecal samples were also taken from the colon of 52 young cattle. The farms of origin of the cattle were not known and hence the feeding practice was also unknown. All faecal samples were transferred into 225 ml of LEB I and cultivated as described below. Meat products The following meat products were collected from supermarkets and butcher's shops: 26 samples of minced meat (mixed pork and beef), 21 samples of different types of fermented raw sausages (pH ranged from 4.8 to 5.4, and aw from 0.932 to 0.890), 15 samples of different " h o t smoked" sausages, and 14 samples of vacuum packaged " h o t smoked" sausages. From the centre of each product 25 g were taken aseptically and transferred into 225 ml of LEB I and cultivated as described below.
175 Carcass meat samples T h e s u r f a c e o f t h e n e c k r e g i o n o f 12 y o u n g c a t t l e c a r c a s s e s w a s s t e r i l i z e d b y burning, and removed. Approximately
25 g o f s a m p l e w a s t h e n a s e p t i c a l l y d r a w n
from the deeper part of the muscle tissue and transferred into 225 ml of LEB I and cultivated as described below. The carcasses had previously been tested by IRN s w a b s a n d w e r e s u b s e q u e n t l y k e p t i n a c h i l l i n g r o o m a t 4 ° C f o r 12 d a y s u n t i l t h e t i m e o f s a m p l i n g . O n l y m e a t s a m p l e s f r o m a n i m a l s w h i c h w e r e s h o w n t o b e L. monocytogenes positive by IRN swabs, were collected.
Cultivation procedure After 24 h incubation at 3 0 ° C , 0.1 ml of LEB I was transferred into 10 ml of LEB II, followed by subculturing on LPM agar, isolation and confirmation as described by Skovgaard and Morgen (1988). This method is essentially the same as that recommended by McClain and Lee (1987). Results
Incidence of Listeria monocytogenes in pigs Results of L. monocytogenes examination in pigs are given in Table I. The overall finding of L. monocytogenes in tonsils was 45%. The incidence of L. monocytogenes TABLE I Presence of Lzsteria spp. in pigs Origin of pigs
Sample
Private farms group I a Private farms group I! b Private farms total
Tonsil swab
State farm A c State farm B d State farms total • b " d
Number of farms (IX>mire)
Number of pigs
L. mnocua Number
%
I.. monocytogenes Number ~,
11 (6)
51
34
66
31
61
25 (9)
52
15
29
15
29
36 (15)
103
49
47
46
45
Faeces
1 (1)
48
7
15
l
2
Faeces
1 (1)
49
38
77
2
4
Faeces
2 (2)
97
45
46
3
3
Tonsil swab Tonsil swab
Group I - - Small farms from the country area where silage feeding of pigs is common. Group II - - Small farms from the country area where s,lage feeding of pigs is seldom. Farm A - - New big farm with good hygtenic conditions; feeding of pigs with dry feed. Farm B - - Old big farm, two-floor system, bad hygmmc condition and wet feeding of pigs (silage).
176 TABLE II Presence of lasterm spp. m young cattle Sample
Number of ammals
l_ mnocua Number
~
L monocytogenes Number %
Faeces IRN a swab Total
52 52 104
20 31 51
38 59 49
10 15 25
19 29 24
IRN: Internal retropharyngeal nodes.
in the tonsillar tissue of pigs from private farms Group I was 61%, whereas the incidence from farms Group II was only 29%. The incidence of L. monocytogenes in faecal samples from 97 pigs originating from state-owned farms was quite low, 3%, but much higher numbers (46%) of the pigs were carriers of other Ltsteria species. The incidence of L. innocua was higher in state farm category B than in state farm category A. The overall finding shows that close to one quarter (24%) of healthy pigs slaughtered in the same slaughterhouse were L. monocytogenes carriers either in tonsils or in faeces, or in both. The strains of Listeria spp. other than L. monocvtogenes were almost all L. innocua, with the exception of three strains of L. welshtmeri and one strain of L. grayi. The latter is, however, no longer accepted as a member of genus Ltsterm (Seeliger and Jones 1986). Inctdence of Listeria monocytogenes m cattle Results of L. monocytogenes findings in cattle are shown in Table II. Among a total of 104 animals, one quarter (24%) were found posttive for L. monocvtogenes, either by the I R N swab technique or by being faecal excretors. L. monocytogenes was demonstrated more frequently in the I R N swabs (29%) than in the faecal samples (19%). The isolated Listeria spp. strains were identified as either L. monocytogenes or L. innocua. lnctdence of Listerta monocytogenes in meat products The findings of L. raonocytogenes in meat and meat products are given in Table III. From Table III it appears that minced meat almost regularly contains L. monocvtogenes and L. innocua, 69% and 80% of samples, respectively, being positive for the two species. Neither of these species were found in the deeper part of the carcass meat of the cattle in spite of the fact that the animals were IRN carriers. Raw fermented sausages, both with small and large diameter, were relatively often contaminated with L. monocytogenes, four of 19 samples, or 19%, being positive.
177 The inner parts of different types of " h o t smoked" sausages (smoked to internal temperatures of 70-75 o C) were free of Listerla spp., but L. monocytogenes were detected in 21% of 14 samples on the surface of the sausages after vacuum packaging, as well as in the liquid accumulation inside the packages, indicating recontamination after the processing.
Discussion
The results show that pigs in Yugoslavia are surprisingly often healthy carriers of L. monocytogenes in the tonsillary tissue. In pigs fed silage, 61% were tonsillary carriers of L. monocytogenes, as compared with 29% positive in pigs from farms using dry feed only. The prevalence of faecal excretors among the pigs vOas 3%, slightly higher than for Danish pigs (Skovgaard and Norrung 1989), but lower than the incidence of 25.6% reported from Hungary (Ralovich, 1984). Since the faecal excretors among pigs did not originate from the same farms as the pigs being tonsillary carriers, no definite answer can be given as to the impact of silage feeding of pigs on the prevalence of L. monocytogenes in tonsillar tissues and the corresponding percentage of animals being faecal excretors. The results indicate, however, that feeding with wet a n d / o r silage feeds contributes to U monocytogenes infections of animals. According to Skovgaard and Norrung (1989), silage feeding of pigs is not normally used in Denmark. This has been used in Yugoslavia for several years. The hygienic condition of the farms may also be of some importance, but firm conclusions on this cannot be drawn from the present investigation. The incidence of faecal excretion in cattle is higher than the figures reported by Dijkstra (1966) and Hofer (1983), who found only 6.7% and 16% positive respectively. The average incidence of L. monocytogenes in young cattle was 24%, the same as in pigs. On the other hand, Skovgaard and Morgen (1988) found a much higher incidence in Danish dairy cows (52%). Unfortunately, no information is available about origin and feeding of the cattle examined in the present investigation, so the differences in incidence cannot be explained by differences in feeding practice. In Denmark wet feeding (silage) of dairy cows is common (Skovgaard and Morgen, 1988), but in Yugoslavia silage feeding is used only to a lesser extent for cattle. From Tables I and II it appears that both cattle and pigs are more often carriers of L. monocytogenes in the tonsillar tissue than they are excretors of the bacteria in faeces. /_. monocytogenes was detected in 45% of the tonsils from pigs, compared with 3% in faeces; and in 29% of the IRN from cattle, compared with 19% in the faecal samples. As proposed by Skovgaard (1988), investigation of tonsils seems to be superior to the faeces examination and is easier to carry out. Further experiments are needed to investigate the relationship between tonsillar carriers and faecal excretors. The findings indicate that pathogenic /_, monocytogenes, when present (in low numbers) in feeds, are able to colonize in the oralpharyngeal lymphatic tissues. On the other hand, low numbers of these bacteria
178
TABLE IIl Presence of l.astena spp. m meat and meat products Sample Meat from carcasses Minced meat
Number examined 12
L mnocua
Number - =
L rnonocytogenes
%
Number
-
-
5[
26
21
80
18
69
21
6
28
4
19
15
-
-
-
14
5
Fermented sausages
•Hot smoked' sausages
packed •hot smoked'
Vacuum
sausages
(surface sample)
35
3
21
= ldsterta not detected.
passing the digestive tract can be suppressed by the competitive microflora, and thus not be detected in faeces. L innocua is usually present in much larger number in feeds than is L monocytogenes, and therefore has a better chance of being detected in faeces of animals than has L monocytogenes. The data shown in Table III indicate that the deep carcass meat is free of Listeria spp. even when the animals are healthy carriers themselves. Secondary surface contamination of meat results in regular findings of L. mono~vtogenes and L. mnocua in minced meat. This is in accordance with results of Lee and McClain (1987), Skovgaard and Morgen (1988), Breuer and Pr~ndl (1988), Schmidt et al. (1988). and Skovgaard and NiSrrung (1989), who found L. monocytogenes in 65-80% of minced meat samples. It is not surprising that L monocytogenes could relatively often (19%) be demonstrated in raw fermented dry sausages. Other authors have found L. monocytogenes in dry sausages at much higher frequencies, ranging from 22% to 83% (Farber, 1987; Nicolas, 1985; Lee and McClain, 1987; Breuer and Pr~indl, 1987; Schmidt et al., 1988). L. monoc.vtogenes is able to survive for a long time during fermentation and storage of such sausages, but seems not be able to grow in them (Trtissel and Jemmi, 1989). In sausages heated to internal temperatures of 70-75 ° C during processing ( ' h o t smoked') we did not find any LJsterta. The surfaces of such sausages can, however, be recontaminated before and during vacuum packaging, and it is therefore not surprising that we detected L. monocytogenes in 21% of the surface samples. Investigations made by Khan et al. (1973), Grau and Vanderlinde (1988), and Glass and Doyle (1989) also showed that L. monocytogenes might be present in vacuum packaged meat and meat products during refrigeration storage and may even be able to multiply under those conditions.
179
L. monocytogenes was shown to be present in 69~ of minced meat samples. In Yugoslavia, minced meat is normally thoroughly cooked, roasted, or grilled before consumption, and L. monocytogenes is likely to be eliminated. Contrary to this, raw fermented sausages are eaten as such, and also vacuum packaged sausages ('hot smoked') are normally consumed without any further heat treatment.
Acknowledgement The author would like to thank Professor Niels Skovgaard for the opportunity to practice the methods of Listeria detection at the Institute of Hygiene and Microbiology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
References Barnes, R., Archer, P., Struck, J. and Istra, G.R. (1989) Listeriosis associated with consumption of turkey franks. MMWR 38, 267-268. Brackett. R.E. (1988) Presence and persistence of lastena monocytogenes in food and water. J. Food. Technol. 42, 162-164. Breuer, J. and Pr~lndl, O. (1988) Nachweis yon Listerien und Vorkommen m Hackfleisch und Mettwiirsten in Osterreich. Arch. Lebensmittelhyg. 39, 28-30. Cottin, J., Genthon. N., Bizon, C. and Carbonnelle, B. (1985) Recherche de Listerla monocytogenes duns des viandes prelev~'es sur 514 bovine. Sci. Ailments. 5, 145-149. Dijkstra, R.G. (1966) Epidemiological investigatsons on Listeriosts in cattle. 3rd Int. Symp. Listenosis, National Insatute for Public Health, Bilthoven, pp. 215-224. Fleming, D.W., Cochi, S.L., MacDonald, K.L. Brondum. J.. Hayes. P.S., Piikayris, B.D., Helmes, M.B., Andurier, A.. Breome, C.V. and Reingold. A.L (1985) Pasteurized milk as a vehicle of infectton m an outbreak of listeriosis. N. Engl. J. Med. 312, 404-407. Farber, J. (1987) quoted from Brackett R.E. (1988). Gilbert, A.J., Miller, K.L. and Roberts, D. (1989) I.~sterm monocvtogenes and chilled foods. Lancet i, 383-384. Glass, K. and Doyle, P. (1989) Fate of L~sterta monocytogenes in processed meat products during refrigerated storage. Appi. Environ. Microblol. 55. 1565-1569. Grau, F.H. and Vanderlinde, P.B. (1988) Growth of L~stena monocytogenes on vacuum packaged beef. Proc. 34th Int. Congr. Meat Science and Technology, Brisbane, 518-519. Gronstol, H. (1979) Listeriosls in sheep, l-asterm monocytogenes excretion and immunological state of healthy sheep. Acta Vet. Scand. 20, 168-179. Hofer, E. (1983) Bacteriologic and epldermologic studses on the occurrence of Listeria monocytogenes in healthy cattle. Zbl. Bakt. Hyg. A 256. 175-183. James. S.M., Fannin, S.L., Agee, B.A., Hall, B., Parker, E., Vogt, J., Run, G., Wilhams, J.. Lieb. L.. Salminen. C., Pendergast, T., Werner, S.B. and Chin, J. (1985) Lstenosis outbreak associated with Mexican style cheese. Calif. Morbid. Mortal. Weekly Rep. 34, 357-359. Kampelmacher, E.H. and Van Noorle Jansen, LM., (1969) Isolation of Ltsteria monocytogenes from faeces of clinically healthy humans and ammals. Zbl. Bakt. Hyg, I. Abt. Orig. 211,353-359. Khan, M.A., Paimas, C.V.. Seaman, A. and Wtxxibme, M., (1973) Survwal versus growth of a facultative psychrotroph in meat and products of meat. Zbl. Bakt. Hyg. I. Abt. Orig. B 157. 277-282. Larsen, E. (1966) Epidemiology of listenosls. The ubiquitous occurrence of Ltsteria monocytogenes. Proc. 3rd Int. Syrup. Listeriosis, National Institute for Pubhc Health. Bilthoven, 295-302. Lee, W.H. and McCiain, D. (1987) quoted from Brackett, R.E. (1988).
180 Lelstner. L., Schrmdt. U. and Kaya, M. (1989) Llstenen bel Fleisch und Fleischerzeugmssen. Mttt. Blatt der BAFF, 28, 1-8. McClam, D. and Lee. W.H. (1987) Isolation and identification of Lasterm monocytogenes from meat U S D A / F S I S / M m r o b t o l . Div., Lab. Comm. No. 57, 9 / 9 / 8 7 . Nicolas. J.A. (1985) Contamination des vlandes et des prodmts de charcuterie par Zasterm monocytogenes des Haute-Vienne. Sci. Aliments. 5, 175-180. Ralowich, B. (1984) Listenosls research - - present situation and perspeeuve. Acadermai Kmdo. Budapest. Schmidt, U., Seehger. H.P.R., Glenn, E., Langner, B. and Letsmer, L. (1988) Llstenenfunde in rohen Flelscherzeugmssen. FlelsChwtrtschaft 10, 1313-1315 Schlech, W.F., Lavique, P.M., Bortolussi, R.A., Allen, A.C., Haldane, A.J., Wort. A.J.. Htghtower, A W.. Johnson, S.E. Kinh, S.H., Nicoils, E.S. and Broome, C.V. (1983). Eptdemic hsteriosts-evldence for transmission by food. N. Engl. J. Med. 308, 203-206. Seeliger, H.P.R. and Jones, D. (1986) G e n u s Lasterla. In: P.H.A. Sheath, N.S. Mair, M.E. Sharpe, J.G. Holt (Eds): Bergey's Manual of Systematic Bacteriology, Vol. 2 Williams & Wilkins, Baltimore. M.D., pp. 1235-1245. Sch~nberg, A. (1988) Z u r aktuellen Sttuation der Listeriose - - eine Ueberslcht. Ber. Miinch. Tier~.rztl. Wschr. 101, 82-84. Skovgaard, N. and Morgen, C.-A. (1988) Detection of Lasterm spp. m faeces from animals, in feeds, and in raw foods of a m m a l origin. Int. J. Food Mlcrobml. 6. 229-242. Skovgaard, N. and Nerrung, B. (1989) The incidence of lasterla spp. in faeces of D a m s h ptgs and in minced pork meat. Int. J. Food Microbml. 8, 59-63. Skovgaard, N. (1988) Personal communication. Trlissel, M. and Jemrm, T. (1989) Das Verhalten yon Ltstena monocytogenes w~hrend der Reifung und Legerung yon kiinstlich kontaminierter Salami und Mettwurst. Fleischwlrtschaft 69, 1586-1592.
173
FOOD 00355
The incidence of Listeria monocytogenes in slaughtered animals, in meat, and in meat products in Yugoslavia Sava Bun6i6 Umverstty of Belgrade, Veterinary Faculty, Department for Meat Hygtene and Technology, Belgrade, Yugoslavia
(Received 7 February 1990: accepted 6 August 1990)
45% of all pigs examined harboured L. monocytogenes m the tonsils, and 3% were faecal excretors. L. monocytogenes was demonstrated m 29% of swabs from the retropharyngeal nodes of cattle and in 19% of faecal samples. The tonsillar and retropharyngeal samples did not correspond to the faecal samples. L. monocytogenes was not demonstrated in the deeper parts of the muscle tissue from 12 beef carcasses all harbouring ~steria in lymph nodes. L. monocytogenes was found in 69% of minced meat (mixed pork and beef) samples. 19% of raw dry sausages and 21% of vacuum-packaged hot smoked sausages were positive for L. monocytogenes. 1_. monocytogenes was not detected in the hot smoked sausages heated to an internal temperature of 70-75°C, after the smokang process. Key words: i.asterta monocytogenes; Swine tonsils; Retropharyngeal nodes; Meat; Meat products
Introduction T h e r e are a n u m b e r of data c o n f i r m i n g the fact that Listeria m o n o c y t o g e n e s is a f o o d - b o r n e p a t h o g e n (Schlech et al., 1983, F l e m i n g et al., 1985; J a m e s et al., 1985; Sch~Snberg, 1988). Implicated foods have been milk a n d dairy products, vegetables, a n d salads. Recently, some i n f o r m a t i o n i n d i c a t i n g that meat a n d p o u l t r y p r o d u c t s c a n also be vectors for h u m a n listeriosis outbreaks, has been reported (Barnes et al., 1989; Leistner et al., 1989; G i l b e r t et al., 1989). H u m a n listeriosis has hitherto not been a notifiable disease in Yugoslavia, a n d no reliable data are available. A n i m a l listeriosis in Yugoslavia has o n l y relatively s e l d o m been reported. Between 1978 a n d 1988 only 24 cases in cattle were registered, 12 in sheep, 1 in a horse, a n d 1 in deer: n o cases of listeriosis have been registered in pigs. F r o m a meat hygiene p o i n t of view, healthy carriers of L. m o n o c y t o g e n e s a m o n g a n i m a l s are i m p o r t a n t as a possible source of c o n t a m i n a t i o n of carcasses in slaughterhouses a n d the processing e n v i r o n m e n t .
Correspondence address: S. Bun~i~. Umversity of Belgrade. Veterinary Faculty, Department for Meat Hygiene and Technology, Bul. JNA 18. 11000 Belgrade, Yugoslavta.
174
I_. monocytogenes has been isolated from the intestinal tract of cattle (Cotin et al.. 1985; Hofer, 1983; Kampelmacher and Van Noorle Jansen, 1969; Skovgaard and Morgen, 1988), sheep (Gr~SnstiSl, 1979), poultry (Larsen, 1966; Skovgaard and Morgen, 1988), and pigs (Larsen, 1966; Skovgaard and NiSrrung, 1989) in different countries. There seem, however, to be no reported data in literature on the prevalence of L. monocytogenes in healthy animals and in meat and meat products in Yugoslavia. The present investigations were initiated to investigate the prevalence of L. monocvtogenes in slaughtered pigs, in young cattle, and in related meat and meat products.
Materials and Methods
Tonsds swabs from pigs Swabs were taken on the slaughter line of a modern slaughterhouse, both from the surface and from fresh cuts of the tonsils from 103 pigs originating from 36 different private farms. The farms represented two groups: in Group I the pigs were fed predominantly on silage, in Group II they were fed dry feed only. The swabs were immediately placed in 10 ml of Listeria Enrichment Broth I (LEB I), followed by subsequent subcultivation in LEB II and isolation and confirmation according to Skovgaard and Morgen (1988) and as described below. Lymph node swabs from cattle Swabs of both the surface and of fresh cuts of the internal retropharyngeal nodes (IRN) were taken from 52 young cattle (bullocks and heifers at the age of 18 months) on the slaughter line of a slaughterhouse. The swabs were placed in 10 ml of LEB I and cultivated as described below. Faecal samples from pigs and cattle Approximately 25 g of faeces were collected on the slaughter line from the lower part of the intestinal tract of 97 pigs originating from two different state-owned farms, A and B. At farm A only dry feed was used, and at farm B silage was also used. Faecal samples were also taken from the colon of 52 young cattle. The farms of origin of the cattle were not known and hence the feeding practice was also unknown. All faecal samples were transferred into 225 ml of LEB I and cultivated as described below. Meat products The following meat products were collected from supermarkets and butcher's shops: 26 samples of minced meat (mixed pork and beef), 21 samples of different types of fermented raw sausages (pH ranged from 4.8 to 5.4, and aw from 0.932 to 0.890), 15 samples of different " h o t smoked" sausages, and 14 samples of vacuum packaged " h o t smoked" sausages. From the centre of each product 25 g were taken aseptically and transferred into 225 ml of LEB I and cultivated as described below.
175 Carcass meat samples T h e s u r f a c e o f t h e n e c k r e g i o n o f 12 y o u n g c a t t l e c a r c a s s e s w a s s t e r i l i z e d b y burning, and removed. Approximately
25 g o f s a m p l e w a s t h e n a s e p t i c a l l y d r a w n
from the deeper part of the muscle tissue and transferred into 225 ml of LEB I and cultivated as described below. The carcasses had previously been tested by IRN s w a b s a n d w e r e s u b s e q u e n t l y k e p t i n a c h i l l i n g r o o m a t 4 ° C f o r 12 d a y s u n t i l t h e t i m e o f s a m p l i n g . O n l y m e a t s a m p l e s f r o m a n i m a l s w h i c h w e r e s h o w n t o b e L. monocytogenes positive by IRN swabs, were collected.
Cultivation procedure After 24 h incubation at 3 0 ° C , 0.1 ml of LEB I was transferred into 10 ml of LEB II, followed by subculturing on LPM agar, isolation and confirmation as described by Skovgaard and Morgen (1988). This method is essentially the same as that recommended by McClain and Lee (1987). Results
Incidence of Listeria monocytogenes in pigs Results of L. monocytogenes examination in pigs are given in Table I. The overall finding of L. monocytogenes in tonsils was 45%. The incidence of L. monocytogenes TABLE I Presence of Lzsteria spp. in pigs Origin of pigs
Sample
Private farms group I a Private farms group I! b Private farms total
Tonsil swab
State farm A c State farm B d State farms total • b " d
Number of farms (IX>mire)
Number of pigs
L. mnocua Number
%
I.. monocytogenes Number ~,
11 (6)
51
34
66
31
61
25 (9)
52
15
29
15
29
36 (15)
103
49
47
46
45
Faeces
1 (1)
48
7
15
l
2
Faeces
1 (1)
49
38
77
2
4
Faeces
2 (2)
97
45
46
3
3
Tonsil swab Tonsil swab
Group I - - Small farms from the country area where silage feeding of pigs is common. Group II - - Small farms from the country area where s,lage feeding of pigs is seldom. Farm A - - New big farm with good hygtenic conditions; feeding of pigs with dry feed. Farm B - - Old big farm, two-floor system, bad hygmmc condition and wet feeding of pigs (silage).
176 TABLE II Presence of lasterm spp. m young cattle Sample
Number of ammals
l_ mnocua Number
~
L monocytogenes Number %
Faeces IRN a swab Total
52 52 104
20 31 51
38 59 49
10 15 25
19 29 24
IRN: Internal retropharyngeal nodes.
in the tonsillar tissue of pigs from private farms Group I was 61%, whereas the incidence from farms Group II was only 29%. The incidence of L. monocytogenes in faecal samples from 97 pigs originating from state-owned farms was quite low, 3%, but much higher numbers (46%) of the pigs were carriers of other Ltsteria species. The incidence of L. innocua was higher in state farm category B than in state farm category A. The overall finding shows that close to one quarter (24%) of healthy pigs slaughtered in the same slaughterhouse were L. monocytogenes carriers either in tonsils or in faeces, or in both. The strains of Listeria spp. other than L. monocvtogenes were almost all L. innocua, with the exception of three strains of L. welshtmeri and one strain of L. grayi. The latter is, however, no longer accepted as a member of genus Ltsterm (Seeliger and Jones 1986). Inctdence of Listeria monocytogenes m cattle Results of L. monocytogenes findings in cattle are shown in Table II. Among a total of 104 animals, one quarter (24%) were found posttive for L. monocvtogenes, either by the I R N swab technique or by being faecal excretors. L. monocytogenes was demonstrated more frequently in the I R N swabs (29%) than in the faecal samples (19%). The isolated Listeria spp. strains were identified as either L. monocytogenes or L. innocua. lnctdence of Listerta monocytogenes in meat products The findings of L. raonocytogenes in meat and meat products are given in Table III. From Table III it appears that minced meat almost regularly contains L. monocvtogenes and L. innocua, 69% and 80% of samples, respectively, being positive for the two species. Neither of these species were found in the deeper part of the carcass meat of the cattle in spite of the fact that the animals were IRN carriers. Raw fermented sausages, both with small and large diameter, were relatively often contaminated with L. monocytogenes, four of 19 samples, or 19%, being positive.
177 The inner parts of different types of " h o t smoked" sausages (smoked to internal temperatures of 70-75 o C) were free of Listerla spp., but L. monocytogenes were detected in 21% of 14 samples on the surface of the sausages after vacuum packaging, as well as in the liquid accumulation inside the packages, indicating recontamination after the processing.
Discussion
The results show that pigs in Yugoslavia are surprisingly often healthy carriers of L. monocytogenes in the tonsillary tissue. In pigs fed silage, 61% were tonsillary carriers of L. monocytogenes, as compared with 29% positive in pigs from farms using dry feed only. The prevalence of faecal excretors among the pigs vOas 3%, slightly higher than for Danish pigs (Skovgaard and Norrung 1989), but lower than the incidence of 25.6% reported from Hungary (Ralovich, 1984). Since the faecal excretors among pigs did not originate from the same farms as the pigs being tonsillary carriers, no definite answer can be given as to the impact of silage feeding of pigs on the prevalence of L. monocytogenes in tonsillar tissues and the corresponding percentage of animals being faecal excretors. The results indicate, however, that feeding with wet a n d / o r silage feeds contributes to U monocytogenes infections of animals. According to Skovgaard and Norrung (1989), silage feeding of pigs is not normally used in Denmark. This has been used in Yugoslavia for several years. The hygienic condition of the farms may also be of some importance, but firm conclusions on this cannot be drawn from the present investigation. The incidence of faecal excretion in cattle is higher than the figures reported by Dijkstra (1966) and Hofer (1983), who found only 6.7% and 16% positive respectively. The average incidence of L. monocytogenes in young cattle was 24%, the same as in pigs. On the other hand, Skovgaard and Morgen (1988) found a much higher incidence in Danish dairy cows (52%). Unfortunately, no information is available about origin and feeding of the cattle examined in the present investigation, so the differences in incidence cannot be explained by differences in feeding practice. In Denmark wet feeding (silage) of dairy cows is common (Skovgaard and Morgen, 1988), but in Yugoslavia silage feeding is used only to a lesser extent for cattle. From Tables I and II it appears that both cattle and pigs are more often carriers of L. monocytogenes in the tonsillar tissue than they are excretors of the bacteria in faeces. /_. monocytogenes was detected in 45% of the tonsils from pigs, compared with 3% in faeces; and in 29% of the IRN from cattle, compared with 19% in the faecal samples. As proposed by Skovgaard (1988), investigation of tonsils seems to be superior to the faeces examination and is easier to carry out. Further experiments are needed to investigate the relationship between tonsillar carriers and faecal excretors. The findings indicate that pathogenic /_, monocytogenes, when present (in low numbers) in feeds, are able to colonize in the oralpharyngeal lymphatic tissues. On the other hand, low numbers of these bacteria
178
TABLE IIl Presence of l.astena spp. m meat and meat products Sample Meat from carcasses Minced meat
Number examined 12
L mnocua
Number - =
L rnonocytogenes
%
Number
-
-
5[
26
21
80
18
69
21
6
28
4
19
15
-
-
-
14
5
Fermented sausages
•Hot smoked' sausages
packed •hot smoked'
Vacuum
sausages
(surface sample)
35
3
21
= ldsterta not detected.
passing the digestive tract can be suppressed by the competitive microflora, and thus not be detected in faeces. L innocua is usually present in much larger number in feeds than is L monocytogenes, and therefore has a better chance of being detected in faeces of animals than has L monocytogenes. The data shown in Table III indicate that the deep carcass meat is free of Listeria spp. even when the animals are healthy carriers themselves. Secondary surface contamination of meat results in regular findings of L. mono~vtogenes and L. mnocua in minced meat. This is in accordance with results of Lee and McClain (1987), Skovgaard and Morgen (1988), Breuer and Pr~ndl (1988), Schmidt et al. (1988). and Skovgaard and NiSrrung (1989), who found L. monocytogenes in 65-80% of minced meat samples. It is not surprising that L monocytogenes could relatively often (19%) be demonstrated in raw fermented dry sausages. Other authors have found L. monocytogenes in dry sausages at much higher frequencies, ranging from 22% to 83% (Farber, 1987; Nicolas, 1985; Lee and McClain, 1987; Breuer and Pr~indl, 1987; Schmidt et al., 1988). L. monoc.vtogenes is able to survive for a long time during fermentation and storage of such sausages, but seems not be able to grow in them (Trtissel and Jemmi, 1989). In sausages heated to internal temperatures of 70-75 ° C during processing ( ' h o t smoked') we did not find any LJsterta. The surfaces of such sausages can, however, be recontaminated before and during vacuum packaging, and it is therefore not surprising that we detected L. monocytogenes in 21% of the surface samples. Investigations made by Khan et al. (1973), Grau and Vanderlinde (1988), and Glass and Doyle (1989) also showed that L. monocytogenes might be present in vacuum packaged meat and meat products during refrigeration storage and may even be able to multiply under those conditions.
179
L. monocytogenes was shown to be present in 69~ of minced meat samples. In Yugoslavia, minced meat is normally thoroughly cooked, roasted, or grilled before consumption, and L. monocytogenes is likely to be eliminated. Contrary to this, raw fermented sausages are eaten as such, and also vacuum packaged sausages ('hot smoked') are normally consumed without any further heat treatment.
Acknowledgement The author would like to thank Professor Niels Skovgaard for the opportunity to practice the methods of Listeria detection at the Institute of Hygiene and Microbiology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
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