- Email: [email protected]
Analysis of bulk goats’ milk and milk-filters from Valtellina and Valchiavenna (Lombardy Prealps) for the presence of Listeria species
Small Ruminant Research 58 (2005) 143–147
Analysis of bulk goats’ milk and milk-filters from Valtellina and Valchiavenna (Lombardy Prealps) for the presence of Listeria species Gabriella Soncini∗ , Luciana Valnegri Dipartimento di Scienze and Tecnologie Veterinarie per la Sicurezza Alimentare, Universit`a di Milano, Via Celoria 10, 20133 Milano, Italy Received 30 March 2004; received in revised form 16 September 2004; accepted 16 September 2004
Abstract Ninety-six samples of bulk untreated goats’ milk and 96 milk-filter samples, obtained monthly from eight farms in Valtellina and Valchiavenna, province of Sondrio, Lombardy Prealps, Italy, from October 2002 to October 2003, were subjected to exhaustive micro-biological analyses for the presence of Listeria spp. Two (2.1%) milk samples and six (6.3%) filter samples were positive for non-pathogenic Listeria species: L. innocua or L. ivanovii in milk; L. innocua, L. ivanovii or L. grayi in milk-filters. No strains pathogenic for humans (in particular L. monocytogenes) were present. This is a reassuring finding particularly as some of the farms are using their untreated milk to produce cheeses on the farm. © 2004 Elsevier B.V. All rights reserved. Keywords: Goats’ milk; Listeria spp.
1. Introduction As emphasised by the WHO in 1988 (Gianfranceschi and Aureli, 2001) Listeria monocytogenes is mainly transmitted to humans via the consumption of contaminated food. The incidence of listeriosis is 2–10 per million (Datta, 2003), but is considerably higher among the newborn (1 per 20,000). Mortality is high (about 28%) (Datta, 2003) perhaps be∗ Corresponding author. Tel.: +39 2 50317872; fax: +39 2 50317870. E-mail address: [email protected] (G. Soncini).
0921-4488/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2004.09.013
cause the illness mainly strikes vulnerable individuals. Despite its low incidence listeriosis has a considerable economic impact, with an annual cost estimated at about 20 million dollars mainly because about 90% of cases require hospitalisation (Tiecco, 2003). L. monocytogenes has been isolated from a large variety of raw and processed foods, including raw and pasteurised milk. Milk products are frequently contaminated and fresh (non-fermented) cheeses, particularly if produced from unpasteurised milk, are a major source of infection (Tiecco, 2003). The consumption of sheep and goats’ milk has been increasing in resent years for several reasons. Astute
// Filter 0 3.1 // 3 No No Mechanical Mechanical
120
130 300
F
G H
Saanen, Camosciata delle Alpi Camosciata delle Alpi Saanen, Camosciata delle Alpi
Local dairy Local dairy
Milk 1.0 1 No Mechanical
0 0 1.0 2.1 1.0 // // 1 2 1 End May–mid Oct No No No Jun–Oct By hand Mechanical By hand Mechanical By hand
Products made on farm Products made on farm Products made on farm Products made on farm Dairy: Nov–May, farm products: June–Oct Local dairy 90 50 90 80 100
Frisa Valtellinese Frisa Valtellinese Camosciata delle Alpi Camosciata delle Alpi Frisa Valtellinese
Percentage of positive samples of each type (milk or filter) from each farm Number of Listeriapositive samples Alpine pasture? Milking method Milk destination
A B C D E
An aliquot (25 ml) was removed from each milk sample under sterile conditions and placed in a sterile Stomacher bag with 225 ml of Palcam listeria selective enrichment broth (Merck-Eurolab, Ovemse, Belgium) followed by incubation at 37 ◦ C for 24 h. Each filter was placed in a sterile Stomacher bag with 225 ml of Palcam broth and also incubated at 37 ◦ C for 24 h. After 24 h, a 1 ml sample of the broth was taken from each bag and placed in a sterile tube with 9 ml of Palcam broth. After incubation at 37 ◦ C for a further 24 h, the
Goats’ breed
3. Microbiological analyses
Number of goats
Samples were taken from the eight establishments whose characteristics are summarised in Table 1. Bulk tank milk samples and a milk-filter were taken monthly from each farm over the period October 2002 to October 2003 (total 96 milk and 96 filter samples). Samples were taken under sterile conditions and kept cool pending analysis.
Table 1 Characteristics of goat farms from which samples were taken and Listeria-positive samples
2. Materials and methods
Sample positive
marketing promotes these products as traditional, at least in Italy (Stella and Cantoni, 2000). They are also increasingly promoted (and used) as part of dietary therapies claimed to promote ossification in the young, to slow bone mass loss in the elderly, and to normalise hematic risk factors for arteriosclerosis (D’Urso, 2000). Goats’ milk in particular is used by people who are allergic to cows’ milk proteins. It is estimated that such allergies affect 1–5% of children and 15–20% adults (D’Urso, 2000). It is noteworthy that sheep and goat milk products are typically consumed by groups (the very young, the elderly, and the sick) who at increased risk for developing severe listeriosis. Furthermore, there is evidence that the rearing and feeding conditions generally employed for sheep and goats put them at greater risk of contracting Listeria infection than cows (Stella and Cantoni, 2000). The aim of the present study was to assess the presence of Listeria spp. in un-pasteurised bulk goats’ milk and milk-filter samples taken monthly from eight farms in the valleys of Valtellina and Valchiavenna of the Lombardy Prealps.
// // Milk Filter Filter
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
Farm
144
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
tube contents were spread (sterile loop) onto two plates: one containing Rapid L. mono (Sanofi-Pasteur, Paris, France) and the other containing Chromogenic listeria agar (OCLA plates, Oxoid, Unipath, Basingstoke, UK). Rapid’L mono is a chromogenic medium sensitive to both the phospholipase C present only in L. monocytogenes and L. ivanovii, and the xylose-fermenting activity present in all Listeria species. L. monocytogenes colonies are blue, L. ivanovii colonies are blue with a yellow halo; colonies of other Listeria are white. The X-glucoside chromogen present in the OCLA medium is cleaved by -glucosidase, common to all listeria, resulting in blue–green coloration. The phospholipase specific for L. monocytogenes and pathogenic L. ivanovii, hydrolyses lecithin in the medium to produce an opaque white halo. Thus L. monocytogenes colonies are blue with an opaque white halo; other Listeria are blue but without the halo. 3.1. Isolation on slants Sufficient numbers of nutrient media slants were prepared for the Listeria-positive species isolated as described above. The slants were incubated at 37 ◦ C for 24 h and then stored in a refrigerator pending identification with the API Listeria and the MicroLog3 systems. 3.2. Identification of isolated colonies with API Listeria system API Listeria (bioM´erieux, Marcy l’Etoile, France) is a miniaturised system in kit form for identifying Listeria species. Basically it consists of a strip containing 10 microtubes, each charged with a different dehydrated substrate for a specific enzyme activity. When Listeria are cultured in the microtubes the products of metabolism lead to colour changes revealed directly or following the addition of a reagent. The pattern of colour changes in the strip of microtubes allows identification of the Listeria species. A sterile loop was used remove samples from young colonies (18–24 h) and transfer them to 2 ml of the supplied API suspension medium to an opacity equal to 1 standard McFarland. Fixed quantities of this suspension were inoculated into each of the 10 microtubes on the strip. The strip was then inserted into the
145
small humid chamber supplied with the kit, closed and incubated aerobically at 36 ± 2 ◦ C for 18–24 h. After adding the ZYM B reagent to the arylamidase (DIM test) microtube, the whole strip was read, referring to the table supplied with the kit, to effect the identification. The percentage probability of the identification was calculated by the software of the identification kit. 3.3. Identification of isolates using the Microlog3 system The Microlog3 system (Biolog, Trust Way, Hayward, CA, USA) is a computerised workstation for identifying microorganisms. The culture is introduced to a variety of preselected carbon sources on the wells of a microplate. After incubation, the pattern of carbon source use, evident from the pattern of colour changes in the wells, is read automatically and compared with a ‘metabolic fingerprint’ database and the result is displayed. Since Listeria are Gram positive, Biolog universal growth (BUG) medium with addition of 5% sheep blood was used to culture each strain isolated as described previously. A sample of each strain was suspended to a specified density in inoculating fluid. One hundred and fifty microliters of this bacterial suspension was pipetted into each well of the microplate and incubated at 37 ◦ C. The plates were read at 6 and 24 h with the Biolog MicroStation and compared to the Gram positive database. Identification was certain if the microorganism was one of the 318 Gram positive species in the database. An identification was suggested by the system if the culture analysed was not one of these species. The Microlog3 software provides a method of calculating both the probability of the identification and the associated confidence interval.
4. Results A total of eight samples (4.2%) were positive for Listeria by the both Rapid’L mono and Chromogenic Listeria systems, six were filter samples and two were milk samples (Table 2). These two systems gave essentially similar results as seen by comparison of Table 3, showing the Rapid’L mono results, and Table 4 showing the Chromogenic Listeria results. Note however that the former method provided species identifications (L.
146
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
Table 2 Results of Rapid’L mono assay and of Chromogenic Listeria assay Farm
Sample
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Rapid’L mono assay
Chromogenic Listeria assay
Appearance of colony
Identification
Appearance of colony
Identification
White Blue with yellow halo White White Blue with yellow halo White White Blue with yellow halo
Listeria spp. Listeria ivanovii Listeria spp. Listeria spp. Listeria ivanovii Listeria spp. Listeria spp. Listeria ivanovii
Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo
Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp.
Table 3 Results of API Listeria assays of morphologically suspicious Listeria colonies isolated by both the Rapid’L mono and Chromogenic Listeria culture systems Farm
Sample
Species identified
Percentage probability of identification
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Listeria innocua Listeria ivanovii Listeria innocua Listeria innocua Listeria ivanovii Listeria innocua Listeria grayi Listeria ivanovii
98 98 99 95 99 97 96 98
monocytogenes and L. ivanovii) while the latter identified only L. monocytogenes. When the Listeria cultures were subjected to species analysis by the API Listeria system (Table 3) and the Microlog3 system (Table 4) again the results were closely similar. In all cases the species identified were the same; the only difference being that the Microlog3 system was able to identify the subspecies in certain cases.
5. Discussion This aim of this study was to assess the extent to which Listeria in general and L. monocytogenes in particular is endemic in dairy goat farms in the valleys of Valtellina and Valchiavenna. We were interested in L. monocytogenes because it is the only pathogen specifically cited by Italian legislation regarding the dairy products sector. Furthermore, it has been shown (Doyle et al., 1987) that for highly contaminated milk (3 × 106 to 2 × 107 cfu/ml) normal pasteurisation (72 ◦ C for 15 s followed by rapid cooling) may not destroy all the organisms if leucocytes are present to harbour and protect them. Thus, a highly contaminated milk batch has the potential to contaminate successive processing stages leading to a contaminated product. Given the ability of L. monocytogenes to multiply at refrigeration temperatures, such a situation could constitute a public health problem. Once it has contaminated milk-processing equipment, L. monocytogenes can form adherent biofilms on
Table 4 Results of Microlog3 system assays of morphologically suspicious Listeria colonies isolated by both the Rapid’L mono and Chromogenic Listeria culture systems Farm
Sample
Species/subspecies identified
Percentage probability of identification with confidence intervals
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Listeria innocua Listeria ivanovii ssp. ivanovii Listeria innocua Listeria innocua Listeria ivanovii ssp. ivanovii Listeria innocua Listeria grayi Listeria ivanovii ssp. londoniensis
99 (0.85–1.82) 97 (0.85–1.75) 85 (0.72–2.15) 92 (0.84–1.98) 95 (0.82–2.75) 99 (0.91–1.26) 100 (0.86–2.06) 72 (0.58–3.00)
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
surfaces, where it may remain vital for long periods and become a source of chronic contamination. Listeria in biofilms can resist cleaning and sanitising procedures, since contact between disinfectant and organism in the lower layer of the biofilm is limited. It has been shown that biofilm Listeria may survive even radical cleaning procedures (Tiecco, 2003). Another reason for concern is that some farms in Italian alpine areas are producing their own cheeses directly from their own untreated milk. This was the case in five of the farms investigated in the present study. Clearly the potential for contamination is greater in such situations. We also expected that farms, which hand-milked their animals (practiced on three of the eight farms studied) would be more prone to Listeria contamination than those, which employed mechanised milking. The results of our study confirmed neither of the above expectations. In fact, the overall results were highly encouraging. We found that L. monocytogenes was absent from all milk and milk-filter samples investigated, although a small proportion of the total samples taken (4.2%) was contaminated with Listeria species that are usually considered non pathogenic for humans (L. innocua, L. ivanovii and L. grayi). Not unexpectedly, we found that more filter samples were Listeria-positive than milk samples. Five of the contaminated filters came from farms with mechanised milking systems incorporating pipelines to transport the milk from the shed to a central collection tank. Such systems were installed in farms with large numbers of milking animals and hence greater probability of Listeria presence. Nevertheless in those establishments with Listeria-positive filters the bulk milk itself was free of contamination, indicating the importance of testing both bulk milk and filters. There is some concern, however, about the presence of L. ivanovii in one milk (1.0%) and two filter (2.1%) samples. Certain strains of this species have been shown to be pathogenic in experimental mice and pregnant sheep (Gualandi, 1995). It is noteworthy that all Listeria-positive samples were obtained in autumn or winter, seasons reported as being associated with increased risk of contamination in relation to a diet of silage or other fermented forage (Gianfranceschi and Aureli, 2001). Studies have
147
shown that milk contamination is favoured not only by such factors as contact with faeces or imperfectly sanitised containers, but that the proportion of animals eliminating Listeria with the faeces increases with the moisture content of the prepared feeds (Gianfranceschi and Aureli, 2001). 6. Conclusion We conclude by emphasising our main finding that no Listeria pathogenic for humans were found in bulk milk and milk-filter samples taken monthly for a year from eight dairy goat farms in the Lombardy Prealps. If these farms are representative, then the goats’ milk and cheese from Italian alpine areas is of reassuringly high quality from the microbiological point of view. Acknowledgements This work was varied out with the support of COFIN 2002 grant from the Italian Ministry of Education, Universities and Research. The authors thank Dr. Deghi, Director of the local dairy (Latteria Sociale di Delebio), and the Sondrio Breeders’ Association (APA di Sondrio) for their help in carrying out this study, and Don Ward for translating the manuscript from Italian and editing it for publication. References Datta, R.A., 2003. Listeria Monocytogenes. International Handbook of Foodborne Pathogens. Marcel Dekker, New York, 105–121. Doyle, M.P., Glass, K.A., Beery, G.T., 1987. Survival of Listeria monocytogenes in milk during high-temperature short time pasteurization. Appl. Environ. Microbiol. 53, 1433–1438. D’Urso, G., 2000. Il latte di capra: specificit`a and qualit`a nutritive. Il Latte (3), 72–77. Gianfranceschi, M., Aureli, P., 2001. Listeria monocytogenes. Recenti sviluppi di igiene and microbiologia degli alimenti. Tecniche Nuove, Milan, 643–662. Gualandi, G., 1995. Listeria Monocytogenes. Trattato Malattie Infettive degli Animali. UTET, Torino, 319-326. Stella, S., Cantoni, C., 2000. Listeriosi nella capra. Obiettivi Documenti Veterinari 9, 69–73. Tiecco, G., 2003. Listeria monocytogenes. Eurocarni 5, 165–175.
Analysis of bulk goats’ milk and milk-filters from Valtellina and Valchiavenna (Lombardy Prealps) for the presence of Listeria species Gabriella Soncini∗ , Luciana Valnegri Dipartimento di Scienze and Tecnologie Veterinarie per la Sicurezza Alimentare, Universit`a di Milano, Via Celoria 10, 20133 Milano, Italy Received 30 March 2004; received in revised form 16 September 2004; accepted 16 September 2004
Abstract Ninety-six samples of bulk untreated goats’ milk and 96 milk-filter samples, obtained monthly from eight farms in Valtellina and Valchiavenna, province of Sondrio, Lombardy Prealps, Italy, from October 2002 to October 2003, were subjected to exhaustive micro-biological analyses for the presence of Listeria spp. Two (2.1%) milk samples and six (6.3%) filter samples were positive for non-pathogenic Listeria species: L. innocua or L. ivanovii in milk; L. innocua, L. ivanovii or L. grayi in milk-filters. No strains pathogenic for humans (in particular L. monocytogenes) were present. This is a reassuring finding particularly as some of the farms are using their untreated milk to produce cheeses on the farm. © 2004 Elsevier B.V. All rights reserved. Keywords: Goats’ milk; Listeria spp.
1. Introduction As emphasised by the WHO in 1988 (Gianfranceschi and Aureli, 2001) Listeria monocytogenes is mainly transmitted to humans via the consumption of contaminated food. The incidence of listeriosis is 2–10 per million (Datta, 2003), but is considerably higher among the newborn (1 per 20,000). Mortality is high (about 28%) (Datta, 2003) perhaps be∗ Corresponding author. Tel.: +39 2 50317872; fax: +39 2 50317870. E-mail address: [email protected] (G. Soncini).
0921-4488/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2004.09.013
cause the illness mainly strikes vulnerable individuals. Despite its low incidence listeriosis has a considerable economic impact, with an annual cost estimated at about 20 million dollars mainly because about 90% of cases require hospitalisation (Tiecco, 2003). L. monocytogenes has been isolated from a large variety of raw and processed foods, including raw and pasteurised milk. Milk products are frequently contaminated and fresh (non-fermented) cheeses, particularly if produced from unpasteurised milk, are a major source of infection (Tiecco, 2003). The consumption of sheep and goats’ milk has been increasing in resent years for several reasons. Astute
// Filter 0 3.1 // 3 No No Mechanical Mechanical
120
130 300
F
G H
Saanen, Camosciata delle Alpi Camosciata delle Alpi Saanen, Camosciata delle Alpi
Local dairy Local dairy
Milk 1.0 1 No Mechanical
0 0 1.0 2.1 1.0 // // 1 2 1 End May–mid Oct No No No Jun–Oct By hand Mechanical By hand Mechanical By hand
Products made on farm Products made on farm Products made on farm Products made on farm Dairy: Nov–May, farm products: June–Oct Local dairy 90 50 90 80 100
Frisa Valtellinese Frisa Valtellinese Camosciata delle Alpi Camosciata delle Alpi Frisa Valtellinese
Percentage of positive samples of each type (milk or filter) from each farm Number of Listeriapositive samples Alpine pasture? Milking method Milk destination
A B C D E
An aliquot (25 ml) was removed from each milk sample under sterile conditions and placed in a sterile Stomacher bag with 225 ml of Palcam listeria selective enrichment broth (Merck-Eurolab, Ovemse, Belgium) followed by incubation at 37 ◦ C for 24 h. Each filter was placed in a sterile Stomacher bag with 225 ml of Palcam broth and also incubated at 37 ◦ C for 24 h. After 24 h, a 1 ml sample of the broth was taken from each bag and placed in a sterile tube with 9 ml of Palcam broth. After incubation at 37 ◦ C for a further 24 h, the
Goats’ breed
3. Microbiological analyses
Number of goats
Samples were taken from the eight establishments whose characteristics are summarised in Table 1. Bulk tank milk samples and a milk-filter were taken monthly from each farm over the period October 2002 to October 2003 (total 96 milk and 96 filter samples). Samples were taken under sterile conditions and kept cool pending analysis.
Table 1 Characteristics of goat farms from which samples were taken and Listeria-positive samples
2. Materials and methods
Sample positive
marketing promotes these products as traditional, at least in Italy (Stella and Cantoni, 2000). They are also increasingly promoted (and used) as part of dietary therapies claimed to promote ossification in the young, to slow bone mass loss in the elderly, and to normalise hematic risk factors for arteriosclerosis (D’Urso, 2000). Goats’ milk in particular is used by people who are allergic to cows’ milk proteins. It is estimated that such allergies affect 1–5% of children and 15–20% adults (D’Urso, 2000). It is noteworthy that sheep and goat milk products are typically consumed by groups (the very young, the elderly, and the sick) who at increased risk for developing severe listeriosis. Furthermore, there is evidence that the rearing and feeding conditions generally employed for sheep and goats put them at greater risk of contracting Listeria infection than cows (Stella and Cantoni, 2000). The aim of the present study was to assess the presence of Listeria spp. in un-pasteurised bulk goats’ milk and milk-filter samples taken monthly from eight farms in the valleys of Valtellina and Valchiavenna of the Lombardy Prealps.
// // Milk Filter Filter
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
Farm
144
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
tube contents were spread (sterile loop) onto two plates: one containing Rapid L. mono (Sanofi-Pasteur, Paris, France) and the other containing Chromogenic listeria agar (OCLA plates, Oxoid, Unipath, Basingstoke, UK). Rapid’L mono is a chromogenic medium sensitive to both the phospholipase C present only in L. monocytogenes and L. ivanovii, and the xylose-fermenting activity present in all Listeria species. L. monocytogenes colonies are blue, L. ivanovii colonies are blue with a yellow halo; colonies of other Listeria are white. The X-glucoside chromogen present in the OCLA medium is cleaved by -glucosidase, common to all listeria, resulting in blue–green coloration. The phospholipase specific for L. monocytogenes and pathogenic L. ivanovii, hydrolyses lecithin in the medium to produce an opaque white halo. Thus L. monocytogenes colonies are blue with an opaque white halo; other Listeria are blue but without the halo. 3.1. Isolation on slants Sufficient numbers of nutrient media slants were prepared for the Listeria-positive species isolated as described above. The slants were incubated at 37 ◦ C for 24 h and then stored in a refrigerator pending identification with the API Listeria and the MicroLog3 systems. 3.2. Identification of isolated colonies with API Listeria system API Listeria (bioM´erieux, Marcy l’Etoile, France) is a miniaturised system in kit form for identifying Listeria species. Basically it consists of a strip containing 10 microtubes, each charged with a different dehydrated substrate for a specific enzyme activity. When Listeria are cultured in the microtubes the products of metabolism lead to colour changes revealed directly or following the addition of a reagent. The pattern of colour changes in the strip of microtubes allows identification of the Listeria species. A sterile loop was used remove samples from young colonies (18–24 h) and transfer them to 2 ml of the supplied API suspension medium to an opacity equal to 1 standard McFarland. Fixed quantities of this suspension were inoculated into each of the 10 microtubes on the strip. The strip was then inserted into the
145
small humid chamber supplied with the kit, closed and incubated aerobically at 36 ± 2 ◦ C for 18–24 h. After adding the ZYM B reagent to the arylamidase (DIM test) microtube, the whole strip was read, referring to the table supplied with the kit, to effect the identification. The percentage probability of the identification was calculated by the software of the identification kit. 3.3. Identification of isolates using the Microlog3 system The Microlog3 system (Biolog, Trust Way, Hayward, CA, USA) is a computerised workstation for identifying microorganisms. The culture is introduced to a variety of preselected carbon sources on the wells of a microplate. After incubation, the pattern of carbon source use, evident from the pattern of colour changes in the wells, is read automatically and compared with a ‘metabolic fingerprint’ database and the result is displayed. Since Listeria are Gram positive, Biolog universal growth (BUG) medium with addition of 5% sheep blood was used to culture each strain isolated as described previously. A sample of each strain was suspended to a specified density in inoculating fluid. One hundred and fifty microliters of this bacterial suspension was pipetted into each well of the microplate and incubated at 37 ◦ C. The plates were read at 6 and 24 h with the Biolog MicroStation and compared to the Gram positive database. Identification was certain if the microorganism was one of the 318 Gram positive species in the database. An identification was suggested by the system if the culture analysed was not one of these species. The Microlog3 software provides a method of calculating both the probability of the identification and the associated confidence interval.
4. Results A total of eight samples (4.2%) were positive for Listeria by the both Rapid’L mono and Chromogenic Listeria systems, six were filter samples and two were milk samples (Table 2). These two systems gave essentially similar results as seen by comparison of Table 3, showing the Rapid’L mono results, and Table 4 showing the Chromogenic Listeria results. Note however that the former method provided species identifications (L.
146
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
Table 2 Results of Rapid’L mono assay and of Chromogenic Listeria assay Farm
Sample
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Rapid’L mono assay
Chromogenic Listeria assay
Appearance of colony
Identification
Appearance of colony
Identification
White Blue with yellow halo White White Blue with yellow halo White White Blue with yellow halo
Listeria spp. Listeria ivanovii Listeria spp. Listeria spp. Listeria ivanovii Listeria spp. Listeria spp. Listeria ivanovii
Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo Blue, no halo
Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp. Listeria spp.
Table 3 Results of API Listeria assays of morphologically suspicious Listeria colonies isolated by both the Rapid’L mono and Chromogenic Listeria culture systems Farm
Sample
Species identified
Percentage probability of identification
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Listeria innocua Listeria ivanovii Listeria innocua Listeria innocua Listeria ivanovii Listeria innocua Listeria grayi Listeria ivanovii
98 98 99 95 99 97 96 98
monocytogenes and L. ivanovii) while the latter identified only L. monocytogenes. When the Listeria cultures were subjected to species analysis by the API Listeria system (Table 3) and the Microlog3 system (Table 4) again the results were closely similar. In all cases the species identified were the same; the only difference being that the Microlog3 system was able to identify the subspecies in certain cases.
5. Discussion This aim of this study was to assess the extent to which Listeria in general and L. monocytogenes in particular is endemic in dairy goat farms in the valleys of Valtellina and Valchiavenna. We were interested in L. monocytogenes because it is the only pathogen specifically cited by Italian legislation regarding the dairy products sector. Furthermore, it has been shown (Doyle et al., 1987) that for highly contaminated milk (3 × 106 to 2 × 107 cfu/ml) normal pasteurisation (72 ◦ C for 15 s followed by rapid cooling) may not destroy all the organisms if leucocytes are present to harbour and protect them. Thus, a highly contaminated milk batch has the potential to contaminate successive processing stages leading to a contaminated product. Given the ability of L. monocytogenes to multiply at refrigeration temperatures, such a situation could constitute a public health problem. Once it has contaminated milk-processing equipment, L. monocytogenes can form adherent biofilms on
Table 4 Results of Microlog3 system assays of morphologically suspicious Listeria colonies isolated by both the Rapid’L mono and Chromogenic Listeria culture systems Farm
Sample
Species/subspecies identified
Percentage probability of identification with confidence intervals
C D D E F H H H
Milk Filter Filter Filter Milk Filter Filter Filter
Listeria innocua Listeria ivanovii ssp. ivanovii Listeria innocua Listeria innocua Listeria ivanovii ssp. ivanovii Listeria innocua Listeria grayi Listeria ivanovii ssp. londoniensis
99 (0.85–1.82) 97 (0.85–1.75) 85 (0.72–2.15) 92 (0.84–1.98) 95 (0.82–2.75) 99 (0.91–1.26) 100 (0.86–2.06) 72 (0.58–3.00)
G. Soncini, L. Valnegri / Small Ruminant Research 58 (2005) 143–147
surfaces, where it may remain vital for long periods and become a source of chronic contamination. Listeria in biofilms can resist cleaning and sanitising procedures, since contact between disinfectant and organism in the lower layer of the biofilm is limited. It has been shown that biofilm Listeria may survive even radical cleaning procedures (Tiecco, 2003). Another reason for concern is that some farms in Italian alpine areas are producing their own cheeses directly from their own untreated milk. This was the case in five of the farms investigated in the present study. Clearly the potential for contamination is greater in such situations. We also expected that farms, which hand-milked their animals (practiced on three of the eight farms studied) would be more prone to Listeria contamination than those, which employed mechanised milking. The results of our study confirmed neither of the above expectations. In fact, the overall results were highly encouraging. We found that L. monocytogenes was absent from all milk and milk-filter samples investigated, although a small proportion of the total samples taken (4.2%) was contaminated with Listeria species that are usually considered non pathogenic for humans (L. innocua, L. ivanovii and L. grayi). Not unexpectedly, we found that more filter samples were Listeria-positive than milk samples. Five of the contaminated filters came from farms with mechanised milking systems incorporating pipelines to transport the milk from the shed to a central collection tank. Such systems were installed in farms with large numbers of milking animals and hence greater probability of Listeria presence. Nevertheless in those establishments with Listeria-positive filters the bulk milk itself was free of contamination, indicating the importance of testing both bulk milk and filters. There is some concern, however, about the presence of L. ivanovii in one milk (1.0%) and two filter (2.1%) samples. Certain strains of this species have been shown to be pathogenic in experimental mice and pregnant sheep (Gualandi, 1995). It is noteworthy that all Listeria-positive samples were obtained in autumn or winter, seasons reported as being associated with increased risk of contamination in relation to a diet of silage or other fermented forage (Gianfranceschi and Aureli, 2001). Studies have
147
shown that milk contamination is favoured not only by such factors as contact with faeces or imperfectly sanitised containers, but that the proportion of animals eliminating Listeria with the faeces increases with the moisture content of the prepared feeds (Gianfranceschi and Aureli, 2001). 6. Conclusion We conclude by emphasising our main finding that no Listeria pathogenic for humans were found in bulk milk and milk-filter samples taken monthly for a year from eight dairy goat farms in the Lombardy Prealps. If these farms are representative, then the goats’ milk and cheese from Italian alpine areas is of reassuringly high quality from the microbiological point of view. Acknowledgements This work was varied out with the support of COFIN 2002 grant from the Italian Ministry of Education, Universities and Research. The authors thank Dr. Deghi, Director of the local dairy (Latteria Sociale di Delebio), and the Sondrio Breeders’ Association (APA di Sondrio) for their help in carrying out this study, and Don Ward for translating the manuscript from Italian and editing it for publication. References Datta, R.A., 2003. Listeria Monocytogenes. International Handbook of Foodborne Pathogens. Marcel Dekker, New York, 105–121. Doyle, M.P., Glass, K.A., Beery, G.T., 1987. Survival of Listeria monocytogenes in milk during high-temperature short time pasteurization. Appl. Environ. Microbiol. 53, 1433–1438. D’Urso, G., 2000. Il latte di capra: specificit`a and qualit`a nutritive. Il Latte (3), 72–77. Gianfranceschi, M., Aureli, P., 2001. Listeria monocytogenes. Recenti sviluppi di igiene and microbiologia degli alimenti. Tecniche Nuove, Milan, 643–662. Gualandi, G., 1995. Listeria Monocytogenes. Trattato Malattie Infettive degli Animali. UTET, Torino, 319-326. Stella, S., Cantoni, C., 2000. Listeriosi nella capra. Obiettivi Documenti Veterinari 9, 69–73. Tiecco, G., 2003. Listeria monocytogenes. Eurocarni 5, 165–175.