J. Vet. Med. B 39, 513-518 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931 - 1793
Departamento de Patologid Animal I . Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
A Direct Plating Method for Monitoring the Contamination of Listeria monocytogenes in silage J. F. FERNANDEZ-GARAYZABAL, M. BLANCO,J. A. VAZQUEZ-BOLAND, V. BRIONES, J. A. GARCIA,C. DELGADO,M. DOMINGO,J. MARCOand L. DOMINGUEZ'" Address of authors: Prof. Dr. LUCASD O M ~ N G URODR~GUEZ, EZ Departamento de Patologia Animal 1. Facultad de Veteriniaria, Universidad Complutense, 28040 Madrid, Spain With 2 figures and one table (Received for publication October 25, 1991)
Summary Twenty-two silage samples were analyzed for the presence of L. monocytogenes using five Listeria selective plating media, with and without previous selective enrichment step. L . monocytogenes was recovered from 3 samples by both procedures, but direct plating allowed the quantification of Listeria population. Two of these positive samples were implicated in outbreaks of listeriosis in sheep; the L. monocytogenes population in these samples was about 106 cells/g. The L . monocytogenes population in the other positive sample was to3 cells/g. Direct isolation of L. monocytogenes was only possible from LPM, PALCAM and LSAMm media. MOX and LSM media were not selective enough to allow direct Listerza isolation. In our hands, LSAMm was the most suitable plating medium for the direct isolation and specific quantification of L. monocytogenes from silage employing a red blood cells overlay technique.
Introduction Reported outbreaks and sporadic cases of listeriosis in ruminants have been increasing 1987). The disease is often associated with the feeding in number since the 60's (RALOVICH, of poor quality silage (GRAY,1960; GRONSTOL, 1979; NICOLAS, 1983; FENSTERBANK et al., 1984; Low and RENTON,1985; FENLON,1986; NICOLAS et al., 1988). Some evidence supports this relationship: most of the cases of listeriosis are detected in flocks fed with this product (BARLOWand MCGORUM,1985; DIJKSTRA,1986); the greater number of cases of listeriosis is found in areas where the silage has an inferior quality (KAMPELMACHER and JANSEN,1979); and the improvement of silage making techniques contributes to a decrease of clinical cases of listeriosis in cattle and sheep (DIJKSTRA,1986). Direct detection and isolation of Listeria from silage has usually been difficult because of the large number of natural microflora. Most of the methods traditionally used for the recovery of Listeria from silage have been basically qualitative, being necessary to use cold 1979; FENSTERBANK et al., 1984; FENLON, or selective enrichment procedures (GRONSTOL, 1986; NICOLAS et al., 1988). Very limited quantitative assessments of Listeria in silage have '' Corresponding author. US. Copyright Clearance Center Code Statement:
0931 - 1793/92/3907-513$02.50/0
514
FERNANDEZ-GARAYZABAL et al.
been done with enrichmet methods using the “most probable number technique”, which is devoided of accuracy (FENLON,1986). U p to date, no data are available on the quantification of L.monocytogenes in silage by direct plating, specially when associated with listeriosis. Although a new generation of highly selective and sensitive Listeria plating media have been developed (BRACKETT et al., 1990), no studies have been done to determine their suitability for the isolation of Listeriu from feedstuffs. In the present work, we have studied the efficiency of the medium and the technique proposed by us for the direct quantification of haemolytic Lzsteriu from foods (BLANCOet al., 1989) compared with four well-known Lzsteriu selective plating media, when they are applied to silage.
Material and Methods Silage samples Twenty-two silage samples (200-3OOg each) were collected from the same number of farms. Each silage sample (a pool of five subsamples taken from different parts of the silo) were placed in sterile plastic bags, brought to the laboratory with refrigeration and analyzed within 24 h.
p H determination O n e tenfold dilution of each silage sample was made in distilled water. The suspension was equilibrated for about 10 minutes and then the p H was measured with a pH-meter (Crison 414).
Plating media The presence of L. monocytogenes was checked by using the following selective plating media: Listeria Selective Agar Medium Modified (LSAMm) (BLANCOet a1.,1989); lithium chloride-phenylethanol-moxalactam (LPM) (LEE and MCCLAIN,1986); polyrnyxin-acriflavine-lithium chlorideceftazidime-aesculin-mannitol (PALCAM) (VAN NETTEN et a1.,1989); Listeria selective medium (LSM) (Oxford formulation) (Oxoid) (CURTISet a1.,1989); and modified Oxford medium (MOX) (LEE, personal communication). The latter medium is an improved modification of the LSM agar which is composed of (1 I): Columbia blood agar base, 39 g; agar, 2 g; aesculin, 1 g; ferric ammonium citrate, 0.5 g; lithium chloride, 15 g; colistin methane sulfonate (Sigma C1511), 10mg; moxalactam (Sigma M1900), 20mg. The ingredients (except moxalactam) are autoclaved (120°C for IOmin). After cooling to about 46 “C, the moxalactam (filter sterilized) is added.
Bacteriological procedures The isolation of Listeria from silage samples was determined simultaneously by direct plating and by a selective enrichment procedure. The enumeration of listeria by direct plating was done as follows: each silage sample (log) was mixed in 90ml of sterile saline solution and homogenized in a stomacher (Colworth 400) for about 3 minutes. This original silage suspension was subsequently tenfold diluted in saline solution. Appropriate dilutions were plated (0.1 ml) by duplicate onto the five selective plating media. Agar plates were always incubated at 37°C for 48 h. Those colonies suspected to be Listeria were counted et al. (1990) as and their haemolysis checked by the overlay technique described by DOMINGUEZ follows: after their incubation, the selective plating media were covered by 1Oml of a red blood cells top-layer. The composition, preparation and storage of the top-layer have been described previously (BLANCOet al., 1989). The plates were reincubated ( 3 0 T for 14h) and finally, the haemolysis was screened. Further identification of the Listeria colonies (three colonies/plate) was based o n the criteria and JONES, 1986) and carried out as described by DOMINGUEZ defined in Bergey’s Manual (SEELIGER et al. (1985). For the selective enrichment, we used a modification of the technique proposed by the Food and et al., 1987). The silage Drug Administration (FDA) to recover L. monocytogenes from foods (LOVETT samples (25 g/each) were added to 225 ml of the FDA enrichment broth (LOVETTet al., 1987), which was incubated at 30 “C and plated by duplicate onto the different selective plating media after 48 h and 7 days of incubation. The identification and haemolytic activity of Listeria were determined as above.
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A Direct Plating Method for Monitoring the Contamination Table 1. Characteristics and population of L. monocytogenes of positive silage samples Sample No
pH
L. monocytogenes populations (log/g) LPM PALCAM LSAMm
Visual qualityb
3' 12" 22
6.0 7.3 8.0
3.2 6.3 6.6
M R
3.3 6.2 6.7
3.3 6.4 6.7
M
L. monocytogenes was isolated after selective enrichment and by direct plating. Direct isolation of L. monocytogenes was not possible on MOX and LSM media. No other Listeria spp. were isolated. M, mouldy; R, rotten. c Silage 3 was contaminated with a single strain (serovar 1/2 a, phagovar 21; 46) of L. monocytogenes. The strains of L. monocytogenes isolated from silage 12 and the brains of sick sheep fed with it had identical markers (serovar 4 b, phagovar 2389; 3552; 2425; 3274; 2671; 52; 47; 108; 340).
Results and Discussion L.monocytogenes was isolated from three out of the twenty-two silages analyzed (numbers three, twelve and twenty two) (Table I), both by direct plating and after 48 h and 7 d of selective enrichment. The recovery and enumeration of L. monocytogenes by direct plating were only possible with LPM, PALCAM and LSAMm, whereas with MOX and LSM it was impossible to recognize any Listeriu colony. The three positive samples were contaminated with high populations of L. monocytogenes (about 106 L. monocytogeneslg of silage in samples twelve and twenty-two and about 103 L. monocytogenes cells/g in sample number three) (Table 1). Although silages are often contaminated simultaneously with more than one species (FENLON,1986), other Listeriu species than L. monocytogenes were not isolated. Silage number three was contaminated with a single strain of L.monocytogenes (serovar 1/2 a, phagovar 21; 46). As far as we know, calves fed with the silage number three did not show any signs of listeriosis. Silages twelve and twenty-two were implicated in two outbreaks of listeriosis in sheep flocks. Only Listerzu colonies isolated from silage twelve were characterized by serotyping and phagetyping; all the tested colonies were L. monocytogenes serovar 4 b and had an identical phagovar (2389; 2552; 2425; 3274; 2671; 52; 47; 108; 340). These markers were also found in the L. monocytogenes colonies isolated from the brains of the sick sheep, confirming the epidemiological relationship between listeriosis outbreaks and silage feeding (VAZQUEZ-BOLAND et al., 1992). During the fermentation of silage, its p H often falls below 4.2, which is inhibitory for Listeria; nevertheless, if the fermentation of silage is not optimal, its p H can be much higher (often between 6.0 and 8.0) than normal (usually < 4.5). Under these conditions, L. monocytogenes can multiply easily, reaching high populations, and increasing, therefore, the probability of its isolation (BLENDEN and SZATALOWICZ, 1967). Seventeen out of the twenty-two silage samples studied had a p H in accordance with an adequate fermentation (pH < 4.5), showed an acceptable visual quality and were Listevia free (data not shown). The other five silage samples had a p H equal to or greater than to 6.0, which is indicative of an improper fermentation; three out of these five samples were contaminated with high populations of L. monocytogenes (Table 1). These results basically agree with the relationship between the p H of silage and the presence of L. monocytogenes reported by other workers. The isolation of L. monocytogenes from silages with p H up to 5.0 is about 30 % higher than from samples with p H lower than this value (GRONSTOL, 1979; FENSTERBANK et al., 1984; FENLON,1986). However, SKOVGAARD and MORGEN(1988) did not show difference in the isolation of Listeriu spp. from silages with low p H (3.4-4.5) and those of higher p H (>4.5). The low performance of MOX and LSM to the direct recovery of L. monocytogenes from silage are related with the low selectivity (about 4.5 logs of decimal reduction of the
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Fig. 1. Direct plating of L. monocytogenes from naturally contaminated silage. It is evident the lower selectivity of LPM, M O X and LSM to inhibit the growth of the silage natural microflora compared with PALCAM and LSAMm. It must be noted that some of the background colonies growth o n LSM and MOX were aesculin positive and resembled Listeria, making the recognition of the real Listeria colonies more complicated
silage natural microflora, data not shown), and to the complicated recognition of the Lixteria colonies on both media (Fig. 1). These results agree with those reported previously in raw milk (DOMINGUEZ et al., 1990). Counts of the L. monocytogenes population in the three contaminated silages were very similar in the other three media (Table 1); however,
Fig. 2. Recognition of the haemolytic colonies of L . monocytogenes isolated from contaminated silagc on LSAMm and PALCAM. The zones of haemolysis around the colonies of L. monocytogenes are sharper and more easily observed on LSAMm (black arrows) than o n PALCAM (white arrows)
A Direct Plating Method for Monitoring the Contamination
517
the best results were obtained when the overlay technique was performed with LSAMm, because the zones of haemolysis produced by the L. monocytogenes colonies were always bigger, sharper and easier to distinguish on this medium (FERNANDEZ-GARAYZABAL et al., 1992) (Fig. 2). Thus, LSAMm was the most suitable selective plating medium for its use, along with the overlay technique, for the quantitative and specific isolation of L. monocytogenes from silage. Although in this study silage samples were only contaminated with L. monocytogenes, it is very likely also a good behaviour of LSAMm in recovering L. monocytogenes from silages contaminated with more than a single Listeriu species. The effectiveness of this medium to discriminate between haemolytic and non-haemolytic colonies of Listeria has been reported by DOMINGUEZ et al. (1990). O u r results show that LSAMm should be a useful tool for monitoring the microbiological quality of silages in veterinary laboratories: i) to quantify the population of haemolytic/pathogenic Listevia in contaminated silages, which would allow to establish a maximum limit of tolerance for these microorganisms (as it has been proposed for human foods), in order to decrease the risk of listeriosis in ruminant flocks; and ii) to confirm the source of infection in clinical cases of listeriosis suspected to be associated with contaminated silage feeding, and to determine the existence of a predominant strain in epidemiological studies of listeriosis outbreaks.
Acknowledgements This work has been supported by the Comision Interministerial de Ciencia y Tecnologia (CfCYT), Reference number ALI90-0554. The authors thank ELI LILLYand GLACO,S. A. who kindly GAMALLO supplied Moxalactam and Ceftazidime respectively. The authors also thank josfi ANTONIO DE JUAN(Asesoria Veterinaria Esla S.A., Mansilla de las Mulas, Leon) who provided the silage samples. We gratefully acknowledge E. ESPAZE(Centre National de Reference de Listeria C. H. R. de Nantes) for serovar determination and J. ROCOURT(Centre International de Reference de Listeria, Institut Pasteur, Paris) for phagovar determination.
References BARLOW,R. M., and B. MCGORUM,1985: Ovine listerial encephalitis: analysis, hypothesis and synthesis. Vet. Rec. 16, 233-236. L. DOMINGUEZ, V. BRIONES, J. A. VAZQUEZ-BOLAND, BLANCO,M., J. F. FERNANDEZ-GARAYZABAL, J.A. GARCIA,and G.SUAREZ,1989: A technique for the direct identification of haemolyticpathogenic listeria on selective plating media. Letters Appl. Microbiol. 9, 125- 128. D. C., and F. T. SZATALOWICZ, 1967: Ecologic aspects of listeriosis. J. Am. Vet. Med. Assoc. BLENDEN, 151, 1761-1766. D. A. GOLDEN, and P. K. CASSIDY, 1990: Assessment of the ability BRACKET,R. E., L. R. BEUCHAT, of plating methods to accurately detect Listeriu in foods. In: Foodborne Listeriosis. Eds. Ed. Elsevier, Amsterdam, Netherlands. MILLER,A. J., J. L. SMITH,and G. A. SAMKUTI. CURTIS,G. D. W., R. G. MITCHELL, G. F. KING, and E. j. GRIFFIN,1989: A selective differential medium for the isolation of Listeriu monocytogenes. Letters Appl. Microbiol. 8, 95-98. DIJKSTRA, R. G., 1986: A fifteen years survey of isolations of Listeriu monocytogenes out of animals and the enivronment in the northern Netherlands. In: Listeriose, Listeria, Listeriosis 1985-1986. Eds. A. L. COURTIEU. Universite de Nantes, France. L., J. A. VAZQUEZ, J. F. FERNANDEZ, P.ECHALECU, E. GOMEZ-LUCIA, E. F. RODRIGUEZ, DOMINGUEZ, and G. SUAREZ, 1985: Microplate technique to determine haemolytic activity for routine typing of Listeriu strains. J. Clin. Microbiol. 24, 99-103. M. BLANCO,V. BRIONES,J. A. VAZQUEZ-BOLAND, DOMINGUEZ, L., J. F. FERNANDEZ-GARAYZABAL, j. L. BLANCO, and G. SUAREZ, 1990: Overlay technique for direct detection and identification of haemolytic Listeriu on selective plating media: comparison of five media. 2. Lebensm. Unters. Forsch. 191, 16-19. D. R., 1986: Rapid quantitative assessment of the distribution of Iisteria in silage implicated FENLON, in a suspected outbreak od listeriosis in calves. Vet. Rec. 118, 240-242. j. GODU,P. GUERRAULT et N. MALO, 1984: Etude des souches de FENSTERBANK, R., A. AUDURIER, Listeriu isolie d'animaux malades et de l'ensilage consomme. Annal. Recher. Vet. 15, 113-118.
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V. BRIONES, FERNANDEZ-GARAYZARAL., J. F., C. DELGADO,M. BLANCO,J. A. VAZQUEZ-BOLAND, G. SUAREZ, and L. DOMINGUEZ, 1992: Role of the potassium tellurite and brain heart infusion in expression of the haemolytic phenotype of Listeriu spp. on agar plates. Appl. Environ. Microbiol. 58, 434-438. GRAY,M. L., 1960: Isolation of Listeriu monocytogenes from oat silage. Science 132, 1767-1768. GRONSTOL, H., 1979: Listeriosis in sheep. Isolation of Listeria monocytogenes from grass silage. Acta Vet. Scand. 20, 492-497. KAMPELMACHER, E. H., and L.M. JANSEN, 1979: Listeriosis in humans and animals in the Netherlands during the past 20 years. In: Proceedings of the 7th International Symposium of Problems of National Agro-Industrial Union Center for Scientific Information, Listeriosis. Eds. I. IVANOV. Bulgaria. LEE,W. H., and D. MCCLAIN, 1986: Improved Listeriu monocytogenes selective agar. Appl. Environ. Microbiol. 52, 1215-1217. LOVETT,J., D. W. FRANCIS,and J. M. HUNT,1987: Listeriu monocytogenes in raw milk: detection, incidence and pathogenicity. J. Food Prot. 50, 188-192. Low, J. C., and C. P. RENTON,1985: Septicemia, encephalitis and abortions in a housed flock of sheep caused by Listeria monocytogenes type 1/2. Vet. Rec. 116, 147-150. NICOLAS, J. A., 1983: RBle de la consommation d’ensilage dans la listeriose ovine. Microbiol. Alimen. Nutrit. 1, 71 -76. M. LAMACHERE, N.VIDAUD, NICOLAS,J.A., E. P. ESPAZE,J. ROCOURT,M. J. CORNUEJOLS, et A. L. COURTIEU, 1988: Listeriose animale et ensilage. IntCrtt de la serotypie et la B. CATIMEL lysotypie dans I’approche Cpidemiologique. Rec. Mid. Vet. 164, 203 -206. RALOVICH, B., 1987: Epidemiology and significance of listeriosis in the European countries. In: Proceedings of the Joint WHO/ROI Consultation on Prevention and Control of Listeriosis. (A. Schonberg, compiled). Institut fur Veterinar-Medizin des Bundes-Gesundheitsamtes (Vet.Med.-Hefte) 5, 21 -55. SEELIGER, H . P. R., and D. JONES,1986: Genus Listeriu Pirie, 1940. In: Bergey’s Manual of Systematic Bacteriology Vol. 2. Eds. SNEATH,H.A., N.S. MAIR, M.E. SHARPE,and J. G. HOLT. Ed. Williams and Wilkins, Baltimore, USA. SKOVGAARD, N., and C. A. MORGEN, 1988: Detection of Listeriu spp. in faeces from animals, in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6, 229-242. G. D . W. CURTIS,and D. A. A. MOSSEL,1989: Liquid and solid selective VANNETTEN,P., I. PERALES, differential media for the detection and enumeration of Listeriu monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8, 299-316. VAZQUEZ-BOLAND, J. A., L. DOMINGUEZ, M. BLANCO, J. ROCOURT, J. F. FERNANDEZ-GARAYZARAL, C. B. GUTIERREZ, R. I. TASCON, and E. F. RODRIGEZ-FERRI, 1992: Epidemiologic investigation of a silage-associated epizootic of ovine listeric encephalitis, using a new Listeriu selective enumeration medium and phage typing. Am. J. Vet. Res. 53, 368-371.
Departamento de Patologid Animal I . Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
A Direct Plating Method for Monitoring the Contamination of Listeria monocytogenes in silage J. F. FERNANDEZ-GARAYZABAL, M. BLANCO,J. A. VAZQUEZ-BOLAND, V. BRIONES, J. A. GARCIA,C. DELGADO,M. DOMINGO,J. MARCOand L. DOMINGUEZ'" Address of authors: Prof. Dr. LUCASD O M ~ N G URODR~GUEZ, EZ Departamento de Patologia Animal 1. Facultad de Veteriniaria, Universidad Complutense, 28040 Madrid, Spain With 2 figures and one table (Received for publication October 25, 1991)
Summary Twenty-two silage samples were analyzed for the presence of L. monocytogenes using five Listeria selective plating media, with and without previous selective enrichment step. L . monocytogenes was recovered from 3 samples by both procedures, but direct plating allowed the quantification of Listeria population. Two of these positive samples were implicated in outbreaks of listeriosis in sheep; the L. monocytogenes population in these samples was about 106 cells/g. The L . monocytogenes population in the other positive sample was to3 cells/g. Direct isolation of L. monocytogenes was only possible from LPM, PALCAM and LSAMm media. MOX and LSM media were not selective enough to allow direct Listerza isolation. In our hands, LSAMm was the most suitable plating medium for the direct isolation and specific quantification of L. monocytogenes from silage employing a red blood cells overlay technique.
Introduction Reported outbreaks and sporadic cases of listeriosis in ruminants have been increasing 1987). The disease is often associated with the feeding in number since the 60's (RALOVICH, of poor quality silage (GRAY,1960; GRONSTOL, 1979; NICOLAS, 1983; FENSTERBANK et al., 1984; Low and RENTON,1985; FENLON,1986; NICOLAS et al., 1988). Some evidence supports this relationship: most of the cases of listeriosis are detected in flocks fed with this product (BARLOWand MCGORUM,1985; DIJKSTRA,1986); the greater number of cases of listeriosis is found in areas where the silage has an inferior quality (KAMPELMACHER and JANSEN,1979); and the improvement of silage making techniques contributes to a decrease of clinical cases of listeriosis in cattle and sheep (DIJKSTRA,1986). Direct detection and isolation of Listeria from silage has usually been difficult because of the large number of natural microflora. Most of the methods traditionally used for the recovery of Listeria from silage have been basically qualitative, being necessary to use cold 1979; FENSTERBANK et al., 1984; FENLON, or selective enrichment procedures (GRONSTOL, 1986; NICOLAS et al., 1988). Very limited quantitative assessments of Listeria in silage have '' Corresponding author. US. Copyright Clearance Center Code Statement:
0931 - 1793/92/3907-513$02.50/0
514
FERNANDEZ-GARAYZABAL et al.
been done with enrichmet methods using the “most probable number technique”, which is devoided of accuracy (FENLON,1986). U p to date, no data are available on the quantification of L.monocytogenes in silage by direct plating, specially when associated with listeriosis. Although a new generation of highly selective and sensitive Listeria plating media have been developed (BRACKETT et al., 1990), no studies have been done to determine their suitability for the isolation of Listeriu from feedstuffs. In the present work, we have studied the efficiency of the medium and the technique proposed by us for the direct quantification of haemolytic Lzsteriu from foods (BLANCOet al., 1989) compared with four well-known Lzsteriu selective plating media, when they are applied to silage.
Material and Methods Silage samples Twenty-two silage samples (200-3OOg each) were collected from the same number of farms. Each silage sample (a pool of five subsamples taken from different parts of the silo) were placed in sterile plastic bags, brought to the laboratory with refrigeration and analyzed within 24 h.
p H determination O n e tenfold dilution of each silage sample was made in distilled water. The suspension was equilibrated for about 10 minutes and then the p H was measured with a pH-meter (Crison 414).
Plating media The presence of L. monocytogenes was checked by using the following selective plating media: Listeria Selective Agar Medium Modified (LSAMm) (BLANCOet a1.,1989); lithium chloride-phenylethanol-moxalactam (LPM) (LEE and MCCLAIN,1986); polyrnyxin-acriflavine-lithium chlorideceftazidime-aesculin-mannitol (PALCAM) (VAN NETTEN et a1.,1989); Listeria selective medium (LSM) (Oxford formulation) (Oxoid) (CURTISet a1.,1989); and modified Oxford medium (MOX) (LEE, personal communication). The latter medium is an improved modification of the LSM agar which is composed of (1 I): Columbia blood agar base, 39 g; agar, 2 g; aesculin, 1 g; ferric ammonium citrate, 0.5 g; lithium chloride, 15 g; colistin methane sulfonate (Sigma C1511), 10mg; moxalactam (Sigma M1900), 20mg. The ingredients (except moxalactam) are autoclaved (120°C for IOmin). After cooling to about 46 “C, the moxalactam (filter sterilized) is added.
Bacteriological procedures The isolation of Listeria from silage samples was determined simultaneously by direct plating and by a selective enrichment procedure. The enumeration of listeria by direct plating was done as follows: each silage sample (log) was mixed in 90ml of sterile saline solution and homogenized in a stomacher (Colworth 400) for about 3 minutes. This original silage suspension was subsequently tenfold diluted in saline solution. Appropriate dilutions were plated (0.1 ml) by duplicate onto the five selective plating media. Agar plates were always incubated at 37°C for 48 h. Those colonies suspected to be Listeria were counted et al. (1990) as and their haemolysis checked by the overlay technique described by DOMINGUEZ follows: after their incubation, the selective plating media were covered by 1Oml of a red blood cells top-layer. The composition, preparation and storage of the top-layer have been described previously (BLANCOet al., 1989). The plates were reincubated ( 3 0 T for 14h) and finally, the haemolysis was screened. Further identification of the Listeria colonies (three colonies/plate) was based o n the criteria and JONES, 1986) and carried out as described by DOMINGUEZ defined in Bergey’s Manual (SEELIGER et al. (1985). For the selective enrichment, we used a modification of the technique proposed by the Food and et al., 1987). The silage Drug Administration (FDA) to recover L. monocytogenes from foods (LOVETT samples (25 g/each) were added to 225 ml of the FDA enrichment broth (LOVETTet al., 1987), which was incubated at 30 “C and plated by duplicate onto the different selective plating media after 48 h and 7 days of incubation. The identification and haemolytic activity of Listeria were determined as above.
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A Direct Plating Method for Monitoring the Contamination Table 1. Characteristics and population of L. monocytogenes of positive silage samples Sample No
pH
L. monocytogenes populations (log/g) LPM PALCAM LSAMm
Visual qualityb
3' 12" 22
6.0 7.3 8.0
3.2 6.3 6.6
M R
3.3 6.2 6.7
3.3 6.4 6.7
M
L. monocytogenes was isolated after selective enrichment and by direct plating. Direct isolation of L. monocytogenes was not possible on MOX and LSM media. No other Listeria spp. were isolated. M, mouldy; R, rotten. c Silage 3 was contaminated with a single strain (serovar 1/2 a, phagovar 21; 46) of L. monocytogenes. The strains of L. monocytogenes isolated from silage 12 and the brains of sick sheep fed with it had identical markers (serovar 4 b, phagovar 2389; 3552; 2425; 3274; 2671; 52; 47; 108; 340).
Results and Discussion L.monocytogenes was isolated from three out of the twenty-two silages analyzed (numbers three, twelve and twenty two) (Table I), both by direct plating and after 48 h and 7 d of selective enrichment. The recovery and enumeration of L. monocytogenes by direct plating were only possible with LPM, PALCAM and LSAMm, whereas with MOX and LSM it was impossible to recognize any Listeriu colony. The three positive samples were contaminated with high populations of L. monocytogenes (about 106 L. monocytogeneslg of silage in samples twelve and twenty-two and about 103 L. monocytogenes cells/g in sample number three) (Table 1). Although silages are often contaminated simultaneously with more than one species (FENLON,1986), other Listeriu species than L. monocytogenes were not isolated. Silage number three was contaminated with a single strain of L.monocytogenes (serovar 1/2 a, phagovar 21; 46). As far as we know, calves fed with the silage number three did not show any signs of listeriosis. Silages twelve and twenty-two were implicated in two outbreaks of listeriosis in sheep flocks. Only Listerzu colonies isolated from silage twelve were characterized by serotyping and phagetyping; all the tested colonies were L. monocytogenes serovar 4 b and had an identical phagovar (2389; 2552; 2425; 3274; 2671; 52; 47; 108; 340). These markers were also found in the L. monocytogenes colonies isolated from the brains of the sick sheep, confirming the epidemiological relationship between listeriosis outbreaks and silage feeding (VAZQUEZ-BOLAND et al., 1992). During the fermentation of silage, its p H often falls below 4.2, which is inhibitory for Listeria; nevertheless, if the fermentation of silage is not optimal, its p H can be much higher (often between 6.0 and 8.0) than normal (usually < 4.5). Under these conditions, L. monocytogenes can multiply easily, reaching high populations, and increasing, therefore, the probability of its isolation (BLENDEN and SZATALOWICZ, 1967). Seventeen out of the twenty-two silage samples studied had a p H in accordance with an adequate fermentation (pH < 4.5), showed an acceptable visual quality and were Listevia free (data not shown). The other five silage samples had a p H equal to or greater than to 6.0, which is indicative of an improper fermentation; three out of these five samples were contaminated with high populations of L. monocytogenes (Table 1). These results basically agree with the relationship between the p H of silage and the presence of L. monocytogenes reported by other workers. The isolation of L. monocytogenes from silages with p H up to 5.0 is about 30 % higher than from samples with p H lower than this value (GRONSTOL, 1979; FENSTERBANK et al., 1984; FENLON,1986). However, SKOVGAARD and MORGEN(1988) did not show difference in the isolation of Listeriu spp. from silages with low p H (3.4-4.5) and those of higher p H (>4.5). The low performance of MOX and LSM to the direct recovery of L. monocytogenes from silage are related with the low selectivity (about 4.5 logs of decimal reduction of the
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Fig. 1. Direct plating of L. monocytogenes from naturally contaminated silage. It is evident the lower selectivity of LPM, M O X and LSM to inhibit the growth of the silage natural microflora compared with PALCAM and LSAMm. It must be noted that some of the background colonies growth o n LSM and MOX were aesculin positive and resembled Listeria, making the recognition of the real Listeria colonies more complicated
silage natural microflora, data not shown), and to the complicated recognition of the Lixteria colonies on both media (Fig. 1). These results agree with those reported previously in raw milk (DOMINGUEZ et al., 1990). Counts of the L. monocytogenes population in the three contaminated silages were very similar in the other three media (Table 1); however,
Fig. 2. Recognition of the haemolytic colonies of L . monocytogenes isolated from contaminated silagc on LSAMm and PALCAM. The zones of haemolysis around the colonies of L. monocytogenes are sharper and more easily observed on LSAMm (black arrows) than o n PALCAM (white arrows)
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the best results were obtained when the overlay technique was performed with LSAMm, because the zones of haemolysis produced by the L. monocytogenes colonies were always bigger, sharper and easier to distinguish on this medium (FERNANDEZ-GARAYZABAL et al., 1992) (Fig. 2). Thus, LSAMm was the most suitable selective plating medium for its use, along with the overlay technique, for the quantitative and specific isolation of L. monocytogenes from silage. Although in this study silage samples were only contaminated with L. monocytogenes, it is very likely also a good behaviour of LSAMm in recovering L. monocytogenes from silages contaminated with more than a single Listeriu species. The effectiveness of this medium to discriminate between haemolytic and non-haemolytic colonies of Listeria has been reported by DOMINGUEZ et al. (1990). O u r results show that LSAMm should be a useful tool for monitoring the microbiological quality of silages in veterinary laboratories: i) to quantify the population of haemolytic/pathogenic Listevia in contaminated silages, which would allow to establish a maximum limit of tolerance for these microorganisms (as it has been proposed for human foods), in order to decrease the risk of listeriosis in ruminant flocks; and ii) to confirm the source of infection in clinical cases of listeriosis suspected to be associated with contaminated silage feeding, and to determine the existence of a predominant strain in epidemiological studies of listeriosis outbreaks.
Acknowledgements This work has been supported by the Comision Interministerial de Ciencia y Tecnologia (CfCYT), Reference number ALI90-0554. The authors thank ELI LILLYand GLACO,S. A. who kindly GAMALLO supplied Moxalactam and Ceftazidime respectively. The authors also thank josfi ANTONIO DE JUAN(Asesoria Veterinaria Esla S.A., Mansilla de las Mulas, Leon) who provided the silage samples. We gratefully acknowledge E. ESPAZE(Centre National de Reference de Listeria C. H. R. de Nantes) for serovar determination and J. ROCOURT(Centre International de Reference de Listeria, Institut Pasteur, Paris) for phagovar determination.
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