Bonardi S.1, Bottarelli A.1, Fusaro S.1, Bentley S.1, Gnappi A.2, Morini A.2
1Istituto di Ispezione degli Alimenti di origine animale - Facoltà di Medicina Veterinaria, Università degli Studi di Parma
2Azienda U. S. L. di Parma - Servizio Veterinario, Distretto Bassa Parmense
Listeria monocytogenes is a foodborne pathogen associated particularly with the consumption of milk, soft cheeses and vegetables. Reports from well-known epidemic outbreaks stress the role of soft cheeses as vehicles of L. monocytogenes; for example, the consumption of Mexican - style soft cheese caused 142 cases of listeriosis in California from January to August 1985 (Linnan et al., 1988), while in Switzerland during the years 1983 to 1987 a large outbreak occurred due to the soft cheese Vacherin Mont d'Or (Bille, 1990). The role of milk as vehicle of the micro-organism was demonstrated in 1983 in Massachusetts, where an epidemic of listeriosis linked to the consumption of pasteurized milk was discovered (Fleming et al., 1985). On that occasion, all human cases of listeriosis were due to pasteurized milk obtained from farms where bovine listeriosis occured at the time of the outbreak. The same phage-type of L. monocitogenes serotype 4b was isolated from both the raw milk of these farms and the human clinical specimens. Since the pasteurization process at the plant had been performed properly at the time of the outbreak, this report suggested the increased heat resistance of intracellular located L. monocytogenes strains. An outbreak of human listeriosis linked to the consumption of coleslaw occurred in the Maritime Province of Canada in 1981 (Schlech et al., 1983). Vehicles of L. monocytogenes transmission were recognised to be the cabbages grown in fields fertilized with raw manure of a flock of sheep where some cases of ovine listeriosis had occurred in the past.
Although the central role of dairy products and vegetables in trasmitting the illness has always been stressed, the microorganism is widespread in the environment and has been isolated from a variety of foods, including raw meat (Carosella, 1990) and meat products (Barbuti et al., 1989; Farber et al., 1989). A survey of about 50 cases of human sporadic listeriosis was carried out in Italy in the years 1985 to 1990 in order to find out the vehicles of infection or the epidemiological associations between illness and sources of the microorganism. Only one case of listeriosic meningitis was considered without a doubt to be of foodborne origin and the vehicle of infection was the raw meat kept in the patient's refrigerator (Casolari et al., 1991). The connection between cases of listeriosis and meat products has been suspected or even demonstrated by a variety of authors such as Schwartz et al. (1988), Cantoni et al. (1989), Kaczmarski and Jones (1989).
Several investigations on the epidemiology of L. monocytogenes in dairy farms have shown a relatively high incidence of intestinal carriers of the microorganism (Skovgaard and Morgen, 1988) and therefore contamination by fecal matter during slaughtering might be responsable for the presence of the pathogen on bovine carcasses.
The present study was carried out to estimate the extent of contamination with Listeria monocytogenes on bovine hides before slaughtering and on carcasses after dehiding. Because of the environmental spreading of the microorganism, different slaughterhouse surfaces and many working instruments were checked for the presence of L. monocytogenes. The fecal matter of slaughtered animals was also analyzed in order to investigate about intestinal carriers of the pathogen.
From September 1996 to May 1997, 625 samples were collected in a 'low-capacity' slaughterhouse (according to Directive 91/497/CEE) in the province of Parma, Italy. Samples were obtained from hides, carcasses, fecal contents, and from the environment like pieces of equipment and working surfaces. For the sampling of hides, bovine carcasses, and environmental surfaces a swab technique was used: an area of 20 cm2 was sampled and the swabs were put into 2 ml of sterile saline solution. Samples from the hides before slaughtering and the bovine carcasses after dehiding were performed on both shoulders and thighs. From slaughtered animals the fecal content was collected under sterile conditions.
The samples were divided into four different groups:
All samples were analyzed according to USDA - FSIS method, based on two subsequent enrichment steps in Listeria Enrichment Broth UVM - 1 and Listeria Enrichment Broth UVM - 2 respectively, followed by plating on the selective media Listeria Selective Agar - Oxford Formulation (McClain and Lee, 1988).
The swabs were transferred into sterile tubes containing 10 ml of Listeria Enrichment Broth UVM - 1 (Oxoid), then incubated for 24 hours at 30° C. Each swab was put into the enrichment broth together with 1 ml of its saline solution. The day after, 0.1 ml of UVM - 1 Broth were transferred to 10 ml of Listeria Enrichment Broth UVM - 2 (Oxoid) and incubated again for 24 hours at 30° C. The solid media Listeria Selective Agar - Oxford Formulation (Merck) was used for plating and all plates were incubated for 48 hours at 35° C. From each agar plates at least five colonies suspected of being Listeria were selected and plated on Columbia Blood Agar with sheep blood (Oxoid). After a 24 hours incubation at 37° C all colonies were Gram - stained, checked for beta hemolysis and tested for biochemical properties using the Microbact 12 L (MedVet) method (Cantoni et al., 1997).
The analytical methods applied to the fecal contents were exactly the same, but for the first enrichment step: 10 g. of fecal matter were mixed with 90 ml of UVM 1 Broth, homogenized and incubated for 24 hours at 30° C. For the second enrichment step 0.1 ml of the UVM 1 Broth were transferred to 10 ml of UVM 2 Broth, as already described.
Listeria spp. strains could be found in 32 of the 625 analysed samples, of which 5 L. monocytogenes (15.6 % of all Listeria isolates), 25 L. innocua, 1 L. seeligeri and 1 L. welshimeri.
From group I samples (512 swabs of 128 bovines before slaughtering and after dehiding) 1 L. monocytogenes and 23 L. innocua strains were isolated (Table 1). Most L. innocua strains were isolated from the hides: 7% of shoulder hides and 5.5% of thigh hides were contaminated by the bacteria (9 and 7 L. innocua strains respectively). L. monocytogenes was never isolated from animals before slaughtering. The carcasses after dehiding gave the following results: 3.1% of shoulders and 2.3 % of thighs were contaminated by L. innocua (4 and 3 L. innocua strains respectively), 0.8% of shoulders were contaminated by L. monocytogenes (1 L. monocytogenes strain).
Table 1: Results of group I samples (512 swabs on bovine hides and carcasses after dehiding)
From group II samples (16 swabs performed on bovine carcasses stored in the chilling room) no Listeria spp. strains were isolated.
Similarly, group III samples (45 swabs performed on both environmental surfaces and working instruments) gave negative results in regard to the isolation of Listeria spp.
From group IV samples (52 bovine faeces) 8 Listeria spp. strains were isolated (Table 2). The isolation rate was 15.4%: 7.7% samples were positive for L. monocytogenes (4 strains), 3.8% for L. innocua (2 strains), 1.9 % for L. seeligeri and 1.9% for L. welshimeri (1 strain each).
Table 2: Results of group IV samples (52 slaughtered bovines' fecal content)
The present study performed at a bovine 'low-capacity' slaughterhouse shows that the contamination by L. monocytogenes and other Listeria spp. was limited to animal hides before slaughtering, to bovine carcasses just after dehiding and to fecal matter, while both the environmental structures and the working instruments resulted Listeria free.
According to these results, there is no doubt that the bacteria were carried into the slaughterhouse by live animals and that both bovine faeces and hides can be considered important vehicles of Listeria spp. The isolation rates decreased from bovine hides ante mortem (16 L. innocua strains isolated from 256 shoulder and thigh swabs, i. e. 6.2% ) to bovine carcasses after dehiding (1 L. monocytogenes and 7 L. innocua strains isolated from 256 shoulder and thigh swabs, i. e. 3.1%). This reduction in the contamination rate demonstrates that well performed slaughtering practice is very important in controlling carcasse bacterial pollution from animal sources. Nevertheless, the most worrying data is the isolation of L. monocytogenes from 1 bovine carcasse just after dehiding. The bacterial strain was isolated from the swab performed on the shoulder, but unfortunately that animal's fecal content was not collected and therefore it is not easy to establish the source of contamination. Vehicles of L. monocytogenes could be the faecal matter of that slaughtered animal or other bovines' faeces or even different environmental sources. Fortunately, the isolation rate of L. monocytogenes from bovine carcasses was rather low, i. e. 0.8%, but the bacteria is able to grow at refrigeration temperatures and therefore the hazard of contaminating near carcasses cannot be ignored.
Bovine carcasses stored in the chilling room did not result to be contaminated by L. monocytogenes or other Listeria species. This important result bears witness to the low incidence of Listeria contamination of carcasses stored in the examined slaughterhouse.
All 45 swabs performed on environmental surfaces (walls and floors of the slaughterhouse and of the chilling room) and on working instuments (knives, cleavers, saws and steel gloves) gave negative results with regard to the isolation of L. monocytogenes and other Listeria species. With no doubt if hygienic measures are respected, as in the examined slaughterhouse, the environmental spread of the microorganisms can be strictly kept under control.
The percentage of cattle carrying Listeria spp. in their fecal matter was 15.4%; a similar epidemiological study carried out in Switzerland by Gobat and Jemmi (1990) pointed out a lower number of intestinal carriers, i. e. 8.3%. The percentage of slaughtered bovines shedding L. monocytogenes in their faeces was 7.7%, as we isolated 4 L. monocytogenes strains from 52 faecal samples. On the contrary, Gobat e Jemmi (1990) did not identify any bovine carrier of L. monocytogenes (0.0%), but Skovgaard and Morgen (1988) found that 39 of 75 samples (52.0%) of bovine faeces collected in different farms contained the microorganism. The shedding rate of L.innocua was 3.8 % (2 isolates of 52 samples) and that of L. seeligeri and L. welshimeri were 1.9% (1 isolate each of 52 samples). Of course the fecal shedding of these bacteria, and especially that of the pathogenic species L. monocytogenes, make their total elimination from a bovine slaughterhouse rather impossible. Nevertheless good hygenic measures, first of all the separation of people working in the 'improper' zone from those working in the 'proper' part of the slaughterhouse, can be of the greatest importance in reducing the risk of contamination. As assessed by Gobat and Jemmi (1990) L. monocytogenes or Listeria spp. can be used as an indicator of germs in a slaughterhouse and can therefore be included in the elaboration of a HACCP program.
The authors thank Mrs. Giuseppina Trentadue and Mrs. Ida Poli for their technical cooperation.
RIASSUNTO. Nel periodo Settembre 1996 - Maggio 1997 in un macello di bovini a capacità limitata della provincia di Parma sono stati prelevati 625 campioni, rappresentati da 512 tamponi eseguiti sulla cute di 128 animali vivi e sulle loro carcasse dopo lo scuoiamento (in corrispondenza delle regioni della spalla e della coscia), 16 tamponi da carcasse stoccate nella cella frigorifera, 45 tamponi ambientali e 52 campioni di materiale fecale, per la ricerca di Listeria spp. ed, in particolare, di L. monocytogenes. Dalla cute dei bovini prima della macellazione la percentuale di tamponi positivi per Listeria spp. è risultata pari al 7% ed al 5,5%, rispettivamente per le regioni della spalla e della coscia; tutti i ceppi isolati appartengono alla specie L. innocua. Dalla carcassa subito dopo lo scuoiamento il 3,9% dei tamponi eseguiti in corrispondenza della spalla ha dato esito positivo per Listeria spp. (0,8% L. monocytogenes e 3,1% L. innocua), mentre dalla regione della coscia la percentuale di isolamento è risultata pari al 2,3% (unica specie isolata: L. innocua). I tamponi eseguiti sulle carcasse stoccate nella cella frigorifera ed i tamponi ambientali hanno dato esito negativo. Dal 15,4% del materiale fecale si sono isolati batteri appartenenti al genere Listeria; in particolare, nel 7,7% dei campioni è stata identificata L. monocytogenes, nel 3,8% L. innocua, nell' 1,9% L. seeligeri e nell' 1,9% L. welshimeri .
SUMMARY. From September 1996 to May 1997, 625 samples were collected in a 'low-capacity' slaughterhouse of cattle in the province of Parma, Italy. The samples were collected as follows: 512 swabs performed on 128 animals, both before slaughtering on shoulder and thigh hides and after dehiding on the same areas of the carcasses; 16 swabs performed on bovine carcasses stored in the chilling room; 45 swabs performed on enviromental surfaces and on working instruments; 52 samples of fecal matter collected from the rectum of slaughtered bovines. All samples were tested for the presence of Listeria spp. and, particularly, L. monocytogenes. The isolation rate of Listeria spp. from the hides before slaughtering was 7% for the swabs performed on the shoulders and 5.5% for those performed on the thighs. All isolated were identified as L. innocua. The isolation rate of Listeria spp. from the carcasses just after dehiding was 3.9% for the swabs performed on the shoulder areas (0.8% L. monocytogenes and 3.1% L. innocua) and 2.3% (all isolates represented by L. innocua) for those collected from the thigh areas. All swabs performed on the carcasses stored in the chilling room and those performed on environmental surfaces and working equipment were negative. The rate of slaughtered bovines shedding Listeria spp. in their faeces was 15.4%; the rate of L. monocytogenes fecal shedding was 7.7%, while 3.8% of slaughtered cattle shedded L. innocua, 1.9% L. seeligeri, and 1.9% L. welshimeri.
ZUSAMMENFASSUNGVon September 1996 bis Mai 1997 haben die Autoren 625 Proben in einem kleinen Rinderschlachthof in der Naehe von Parma (Italien) entgenommen, um die Verbreitung von Listeria spp. - und besonders von L. monocytogenes - zu untersuchen. Die Autoren haben 512 Wattebaeusche von 128 Rindviehren ante-mortem und post-mortem abgerieben. Besonders wurden die Wattebaeusche je Rindvieh von der Schulterhaut und der Oberschenkelhaut abgerieben. Nach dem Schlachten und dem Abhaeuten wurden die Proben von den entsprechenden Flaechen genommen. Es wurden ausserdem 16 Wattebaeusche von ebensovielen Rinderhaelften, die sich in der Kuehlzelle befanden, abgerieben. Von einigen Schlachthofflaechen und Schlachtwerkzeugen wurden 45 Wattebaeusche abgerieben. Die Autoren haben ausserdem 52 Faekalienproben von ebensovielen geschlachten Rindviehen genommen.
Die Ergebnisse unserer Untersuchung werden als Isolierungprozentual ausgedrueckt:
- n 7% Listeria spp. von den Schulterhautwattebaeuschen (alle L. innocua Staemme)
- n 5,5% Listeria spp. von den Oberschenkelhautwattebaeuschen (alle L. innocua Staemme)
- n 3,9% Listeria spp. von den Schulterwattebaeuschen nach dem Schlachten und dem Abhaeuten (0,8% L. monocytogenes und 3,1% L. innocua )
- n 2,3% Listeria spp. von den Oberschenkelwattebaeuschen nach dem Schlachten und dem Abhaeuten (alle L. innocua Staemme)
- n 15,4% Listeria spp. von den Faekalienproben (7,7% L. monocytogenes, 3,8% L. innocua, 1,9% L. seeligeri und 1,9% L. welshimeri)
Keinen Listeriastamm haben die Autoren von den anderen Wattebaeuschen isoliert.
Keywords: Listeria spp., Listeria monocytogenes, slaughterhouse, bovine carcasses, bovine faeces
Parole chiave: Listeria spp., Listeria monocytogenes, macello, carcasse bovine, feci bovine
Schluesselwoerter: Listeria spp., Listeria monocytogenes, Schlachthof, Rinderhaelften, Rinderfaekalien
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