HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Muehlherr, J. E. Articles by Stephan, R. Search for Related Content PubMed PubMed Citation Articles by Muehlherr, J. E. Articles by Stephan, R.

Microbiological Quality of Raw Goat’s and Ewe’s Bulk-Tank Milk in Switzerland

J. E. Muehlherr^*, C. Zweifel^*, S. Corti^*, J. E. Blanco and R. Stephan^*

^* Institute for Food Safety and Hygiene, Faculty of Veterinary Medicine, University of Zurich, CH-8057 Zurich, Switzerland
Laboratorio de Referencia de E. coli (LREC), Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain

Corresponding author: R. Stephan; e-mail stephanr{at}fsafety.unizh.ch.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
A total of 407 samples of bulk-tank milk (344 of goat’s milk and 63 of ewe’s milk) collected from 403 different farms throughout Switzerland, was examined. The number of farms investigated in this study represents 8% of the country’s dairy-goat and 15% of its dairy-sheep farms. Standard plate counts and Enterobacteriaceae counts were performed on each sample. Furthermore, the prevalence of Staphylococcus aureus, Campylobacter spp., Shiga toxin-producing Escherichia coli, Salmonella spp., and Mycobacterium avium ssp. paratuberculosis was studied. The median standard plate count for bulk-tank milk from small ruminants was 4.70 log cfu/ml (4.69 log cfu/ml for goat’s milk and 4.78 log cfu/ml for ewe’s milk), with a minimum of 2.00 log cfu/ml and a maximum of 8.64 log cfu/ml. Enterobacteriaceae were detected in 212 (61.6%) goat’s milk and 45 (71.4%) ewe’s milk samples, whereas S. aureus was detected in 109 (31.7%) samples of goat’s milk and 21 (33.3%) samples of ewe’s milk.

Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. However, 16.3% of the goat’s milk and 12.7% of the ewe’s milk samples were polymerase chain reaction (PCR)-positive for Shiga toxin-producing E. coli. Seventy-nine (23.0%) goat’s tank-milk and 15 (23.8%) ewe’s tank-milk samples were PCR-positive for insertion sequence 900, providing presumptive evidence for the presence of M. avium ssp. paratuberculosis. These results form the basis for determining the microbiological quality standards for goat’s and ewe’s milk. Moreover, the data presented form part of the risk assessment program for raw milk from small ruminants in Switzerland.

Key Words: food-borne pathogen • goat’s and ewe’s milk • microbiological quality • prevalence

Abbreviation key: CI = confidence interval, IS = insertion sequence, MAP = Mycobacterium avium ssp. paratuberculosis, SPC = standard plate count, STEC = Shiga toxin-producing Escherichia coli, D-value = decimal reduction time

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Public health problems associated with consumption of unpasteurized cow’s milk and raw-milk products have been well documented (Barrett, 1986; Keene et al., 1997; Cody et al., 1999; De Valk et al., 2000; Kalman et al., 2000; De Buyser et al., 2001; Harrington et al., 2002). There is no evidence that the risk from unpasteurized goat’s or ewe’s milk is any lower (Hutchinson et al., 1985; Allerberger et al., 2001; McIntyre et al., 2002). Pathogenic microorganisms can gain access to milk either by fecal contamination or by direct excretion from the udder into the milk.

Unlike cow’s milk, which is subject to stringent hygiene and quality regulations, microbiological standards for production and distribution of goat’s and ewe’s milk are more relaxed in Switzerland and are not subject to specific microbiological standards in a legal sense. Furthermore, the literature provides only limited data on the microbiology of goat’s and ewe’s milk in other countries (Roberts, 1985; Little and Louvois, 1999; Foschino et al., 2002) and none at all for Switzerland.

Nevertheless, there is growing demand for unpasteurized goat’s and ewe’s milk by consumers in Switzerland. This is due to the increasing number of children suffering from intolerance to cow’s milk as well as to the demand for natural and unprocessed food. Furthermore, traditional Swiss goat’s and sheep’s cheese is typically made from raw milk with the natural microflora responsible for enhancing desirable flavor characteristics of the final product. So there is a clear need to find out more about the present situation regarding the quality of goat’s and ewe’s milk in Switzerland.

The objectives of this study were to 1) determine the microbiological status of goat’s and ewe’s milk in Switzerland and 2) study the prevalence of food-borne pathogens, especially Staphylococcus aureus, Campylobacter spp., Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Mycobacterium avium ssp. paratuberculosis (MAP).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Milk Samples
Between February and August 2002, a total of 407 bulk-tank milk samples (344 goat’s milk and 63 ewe’s milk) were collected from 403 farms with a total of approximately 11,000 animals (8000 goats and 3000 ewes). The number of farms investigated represents 8% of Switzerland’s dairy-goat and 15% of its dairy-sheep farms. Goat farms ranged in size from two to 180 animals and ewe farms from 2 to 300 animals. The farms were distributed throughout 25 of Switzerland’s 26 cantons (Figure 1) and were categorized in three groups (<6 animals, 6 to 25 animals, >25 animals). The numbers of farms investigated in these groups were selected according to the frequency of distribution of the various farm sizes in Switzerland.

View larger version (30K): [in this window] [in a new window]   Figure 1. Sampling of small ruminant’s milk throughout 25 of Switzerland’s 26 cantons (goat’s bulk-tank milks/ewe’s bulk-tank milks).

Standard Plate Count and Enterobacteriaceae Count
Standard plate count (SPC) and Enterobacteriaceae counts were determined by pour plate method with plate count agar (Oxoid, Basingstoke, U.K.) and violet-red bile glucose agar (BBL, Cockeysville, MD), respectively. The detection limit was at 1.0 x 10⁰ cfu/ml.

Staphylococcus aureus
Numbers of S. aureus were determined by surface-plating the samples on rabbit-plasma-fibrinogen agar (Oxoid CM 275 with RPF supplement Sedia 2000-0100). The detection limit was 1.0 x 10¹ cfu/ml. The number of coagulase-positive staphylococci was obtained by direct enumeration of typical colonies (tellurite-positive colonies with an opaque halo).

Campylobacter spp.
Ten milliliters of milk was inoculated into 100 ml of selective enrichment broth (Brucella Bouillon [Difco 0495-17-3, Becton Dickinson, Sparks, MD] with Campylobacter growth supplement [Oxoid SR84] and Skirrow Campylobacter selective supplement [Oxoid SR69]) and incubated at 42°C for 48 h under microaerobic conditions provided by commercial gas packs (CampyGen from Oxoid). The enrichment samples were then streaked onto selective agar media (Brucella agar, [Difco 0964-17-5, Becton Dickinson] with 6% horse blood [Oxoid SR48], as well as Butzler Campylobacter selective supplement [Oxoid SR85]) and incubated at 42°C for another 48 h under microaerobic conditions. Suspicious colonies, appearing translucent white, moist, and glistening, were selected for further identification.

Shiga Toxin-Producing E. coli
From each sample, 25 ml was enriched in 225 ml of brilliant-green bile broth (BBL) at 37°C for 24 h. The enrichment broth was then evaluated by PCR with primers based on sequences targeting a region between stx1 and stx2 genes (Burnens et al., 1995). Bacterial DNA was prepared by incubating 2 µl of brilliant-green bile broth in 42 µl of double-distilled water at 100°C for 10 min. Amplifications were carried out with a total volume of 50 µl containing 200 µM of dNTP, 30 pmol of each primer, 5 µl of 10-fold concentrated polymerase synthesis buffer, and 2.5 U of Taq-DNA-polymerase (Promega, Madison, WI) in a Biometra DNA cycler. The amplified products were visualized by gel electrophoresis on 0.9% agarose gel stained with ethidium bromide.

From 40 PCR-positive samples, the enrichment broth was plated onto sheep blood agar (Trypticase-Soy-Agar, BBL with 5% sheep blood), and five single colonies were tested by the same PCR protocol to obtain STEC isolates. The strains were identified with classic biochemical tests (acid production from mannitol, o-nitrophenyl-ß-D-galactopyranoside test, H₂S and indole production, as well as proof of urease and lysine decarboxylase). All strains were examined for sorbitol fermentation, for ß-D-glucuronidase activity, and for the presence of stx1 and stx2 genes (Blanco et al., 2003). The PCR-RFLP was used for subtyping stx2 variants (Piérard et al., 1998). Furthermore, PCR was employed to determine the presence of the eae, hlyA, and astA genes (Schmidt et al., 1994; 1995; Yamamoto and Nakazawa, 1997).

The determination of O and H antigens was carried out by the method described by Guinée et al. (1981), employing all available O (O1-O181) and H (H1-H56) antisera.

Salmonella spp.
Salmonella spp. were detected in a two-stage enrichment procedure. Twenty-five ml of milk was pre-enriched in 225 ml of buffered peptone water (Oxoid CM 509) at 37°C for 24 h. Ten milliliters of the pre-enrichment sample was then incubated in 100 ml of tetrathionate (Oxoid CM 343) or 0.1 ml of the pre-enriched sample in 10 ml of Rappaport-Vassiliadis medium (Oxoid CM 669) at 37°C or 43°C for another 24 h. Enrichments were then streaked onto brilliant-green phenol red agar (Difco 0285-17-7, Becton Dickinson) and mannitol-lysin crystal-violet brilliant green agar (Brandenberger, Zurich, Switzerland). Both selective media were incubated at 37°C for 24 h.

Mycobacterium avium ssp. paratuberculosis
Ten milliliters of each sample was mixed with 100 µl of Triton X-100 (Calbiochem, Darmstadt, Germany) and centrifuged at 4500 rpm for 30 min. Afterwards, the pellet was transferred to a Blue Ribolyser tube (Hybaid, Ashford, U.K.), resuspended in 400 µl of mycobacterial lysis buffer (EDTA, 2mM; sodium chloride, 400 mM, TrisHCl, 10 mM (pH 8.0); 0.6% SDS; proteinase K, 33 µl/ml) and incubated overnight at 37°C. For access to target DNA, samples were centrifuged at 6.5 m/s for 45 s in a RiboLyser (Hybaid). The DNA was then extracted with 1 volume of phenol/chloroform/isoamyl-alcohol (25:24:1) and precipitated with 3 M potassium acetate for 30 min at -70°C, centrifuged (13,000 rpm, 15 min), washed in 70% ethanol, dried, and resuspended in TE-buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Five microliters of the resuspended DNA was used for an insertion sequence (IS) 900-nested PCR specific to MAP. According to Hermon-Taylor et al. (2000), the primers p90 5'-GAA GGG TGT TCG GGG CCG TCG CTT AGG-3' and p91 5'-GGC GTT GAG GTC GAT CGC CCA CGT GAC-3' were used for the first 30 amplification cycles. The PCR mix consisted of 50 µl of reaction volume containing a final concentration of 2 µM of each primer, 2.5 mM MgCl₂, 100 µM dNTP, and 2 U Taq polymerase (Promega) in 1x reaction buffer (Promega). The reactions were cycled as follows: initial denaturation at 94°C for 5 min; 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 3 min; and a final extension of 72°C for 7 min.

Two microliters from the primary amplification were then used with the primers AV1 5'-ATG TGG TTG CTG TGT TGG ATG G-3' and AV2 5'-CCG CCG CAA TCA ACT CCA G-3' for the nested PCR. The PCR mix consisted of a 50-µl reaction volume containing a final concentration of 2 µM of each primer, 1.5 mM MgCl₂, 100 µM dNTP, and 2 U Taq polymerase (Promega) in 1x reaction buffer (Promega). The reactions were cycled as follows: initial denaturation at 94°C for 5 min; 40 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 3 min; and a final extension of 72°C for 7 min. The PCR products were visualized on 1.5% agrose gels (Eurobio, Les Ulis Cedex, France). The IS900-nested product is 298 bp in length.

Statistical Analysis
The statistical significance was determined by the ²-test (P < 0.05), the Mann-Whitney U-test (P < 0.05) and the Kruskal Wallis H-test (P < 0.05).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The median SPC for bulk-tank milk from small ruminants was 4.70 log cfu/ml, with a minimum of 2.00 log cfu/ml and a maximum of 8.64 log cfu/ml. Median SPC was 4.69 log cfu/ml for goat’s milk and 4.78 log cfu/ml for ewe’s milk. Enterobacteriaceae were detected in 212 (61.6%; confidence interval [CI] of 95% = 61.01 to 71.96) goat’s milk and 45 (71.4%, CI 95% = 56.98 to 80.77) ewe’s milk samples. The median value was 1.88 log cfu/ml for goat’s milk, with a maximum of 7.64 log cfu/ml, and 2.08 log cfu/ml for ewe’s milk, with a maximum of 5.34 log cfu/ml. Selected statistical values of SPC and Enterobacteriaceae counts are summarized in Table 1. The frequency distributions of SPC and Enterobacteriaceae counts for goat’s and ewe’s milk are shown in Figure 2.

View larger version (29K): [in this window] [in a new window]   Figure 2. Frequency distributions of SPC and Enterobacteriaceae counts for goat’s and ewe’s bulk-tank milk.

The prevalence of Campylobacter spp., STEC, Salmonella spp., and MAP are shown in Table 3. Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. However, STEC PCR was positive in 62 (15.2%) of the 407 milk samples examined. Also, 16.3% (CI 95% = 15.63 to 24.99) of goat’s milk samples and 12.7% (CI 95% = 5.65 to 23.50) of ewe’s milk samples were stx positive. Twelve STEC strains were isolated and further characterized (Table 4). All the strains belonged to the non-O157 E. coli group and tested positive for sorbitol and ß-D-glucuronidase. One strain harbored only the stx1 gene, one strain only the stx2 gene, and 10 strains the stx1 and stx2 genes. Nine strains tested positive for EHEC-hlyA, one strain tested positive for the eae gene, and no strain harbored the astA gene.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
No significant differences could be detected in SPC and Enterobacteriaceae counts as a function of the animal species (sheep/goats). Therefore, the same microbiological limit value can be used to assess the quality of both sheep’s and goat’s milk. According to European Council Directive 92/46/EEC, raw goat’s or sheep’s milk used to produce drinking milk and products from heat-treated milk must not contain more than 6.00 log cfu/ml (SPC), and the milk used for the manufacture of raw-milk products not more than 5.70 log cfu/ml (SPC). In our study, 77% of the goat’s milk and 86% of the sheep’s milk or 73% of the goat’s and 81% of the sheep’s milk SPC results, respectively, were below the SPC limits fixed by the EU regulations. The microbiological quality standard stipulated by European Council Directive 92/46/EEC may therefore be used as a basis for developing a specific quality standard system for milk from small ruminants in Switzerland.

The delivery frequency of the milk supplied had an impact on the SPC of the milk samples. The observed significant differences in SPC between farms with daily milk deliveries and those with deliveries every second or third day suggested some difficulty in cooling the milk on farm for a longer period. The increase in microbial contamination of milk samples obtained from farms with more animals was quite unexpected. We assumed that farms with more animals would have more professional hygiene management, resulting in lower microbial counts.

The SPC median values of goat’s and ewe’s milk were one log unit higher than the median value of cow’s tank milk (Stephan and Bühler, 2001). In addition, striking differences were found in the prevalence of the Enterobacteriaceae: 7% for cow’s milk samples compared with 62% for goat’s milk and 70% for sheep’s milk samples. These results suggest some difficulty in managing the hygiene quality of small ruminant’s milk, which may be due to a series of factors including the milking system. In contrast, the prevalence of S. aureus in milk samples from small ruminants (32%) is only half as frequent as that found in cow’s milk samples (Stephan et al., 2002).

Other surveys of pathogen prevalence in goat’s and ewe’s milk provided results comparable to those of our study, showing no Salmonella or Campylobacter-positive samples (Little et al., 1999; Foschino et al., 2002; Morgan et al., 2003). However, 16.3% of the goat’s milk and 12.7% of the ewe’s milk samples were positive for STEC. The importance of the STEC group has increased since the first description of a food-borne infection caused by E. coli O157:H7 (Riley et al., 1983). Human STEC infection after raw milk consumption was first mentioned by Martin et al. (1986) and has now gained greater importance, in particular due to the very low minimum infectious dose. The risk associated with consumption of unpasteurized goat’s milk is documented in the literature (Bielaszewska et al., 1997; McIntyre et al., 2002). Frequently, STEC strains isolated from patients are of serotype O157 and show a typical virulence spectrum, with such strains tending to be stx2 and eae-positive (Boerlin et al., 1999). The stx2d variant was the principal stx2 subtype in strains isolated from small ruminants in our study, and has so far not been detected in STEC strains isolated from patients. Only one STEC strain with serotype ONT:H19 harbored stx2 and eae genes. Despite the low prevalence of strains with the typical virulence pattern in bulk-tank samples, infections due to consumption of raw milk can not be excluded. However, pasteurization of milk (71.7°C for 15 s, Swiss food regulation) offers sufficient protection.

MAP is a pathogen that causes chronic, granulomatous inflammation of the intestine in various animals, including cattle and small ruminants, and can also be cultured from milk of apparently healthy animals (Streeter et al., 1995). Today, it seems increasingly probable that MAP could play a role in the etiology of Crohn’s disease (Hermon-Taylor et al., 2000). However, conclusive evidence for the etiological role of MAP in Crohn’s disease is still lacking.

Raw milk may be a potential vehicle for transmission of MAP to humans. Therefore, MAP has acquired special relevance to food hygiene. Moreover, results of laboratory pasteurization tests of raw whole milk spiked with MAP showed that MAP was capable of surviving pasteurization (Chiodini and Hermon-Taylor, 1993; Grant et al., 1998; Keswani and Frank, 1998; Sung and Collins, 1998). Nevertheless, conflicting results can be found in the literature. Sung and Collins (1998) reported a decimal reduction time (D-value) for MAP in milk of 11 s at 71°C, indicating that MAP could survive HTST pasteurization if the initial numbers were >1 log cfu/ml milk. Pearce et al. (2001), however, reported a D-value of 2.03 s at 72°C, which is equivalent to a >7 log reduction at 72°C for 15 s (HTST pasteurization). Grant et al. (2002) pasteurized raw cows’ milk naturally infected with MAP and found that MAP survived commercial-scale pasteurization at 73°C for 15 s and 25 s with and without prior homogenization if the bacterial cells were present in sufficient numbers before heat treatment.

The 23% prevalence in goat’s and 24% in ewe’s milk IS900 PCR-positive bulk-milk samples indicates a wide distribution of subclinical MAP infections in small dairy ruminants in Switzerland. Polymerase chain reaction targeting the 5' end of IS900 has been considered specific for identification of MAP and is frequently applied to confirm the presence of this organism. However, the finding of an insertion sequence very similar to IS900 in a Mycobacterium sp. unrelated to MAP has raised questions concerning the reliability of PCR methods for detection of MAP (Cousins et al., 1999; Englund et al., 2002).

No other comparative data on the occurrence of MAP in goat’s and ewe’s milk are currently available for Switzerland. Djonne et al. (2003) examined raw goat’s milk in Norway. In their study, 7.1% of the samples tested positive by an immunomagnetic separation PCR method. In a survey by Grant et al. (2001) in England, one raw goat-milk sample from 104 raw sheep’s and goat’s milk samples tested positive for the presence of MAP by immunomagnetic separation PCR.

In a previous study, we demonstrated that 19.7% of raw bulk-tank milk samples obtained from cows in Switzerland were IS900 positive (Corti and Stephan, 2002). Compared with cow’s milk, no statistically significant regional differences in MAP herd-level prevalence were found for small ruminants.

The results of this study could form the basis for determining specific microbiological quality standards for goat’s and ewe’s milk in Switzerland. Moreover, they constitute part of the Swiss risk assessment program for raw milk from small ruminants.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors would like to thank F. Del Chicca and S. Schumacher for their technical assistance. This study was partly supported by a grant from the Fondo de Investigación Sanitaria (FIS G03/025-COLIRED-157).

Received for publication March 12, 2003. Accepted for publication June 5, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 


Allerberger, F., M. Wagner, P. Schweiger, H. P. Rammer, A. Resch, M. P. Dierich, A.W. Friedrich, and H. Karch. 2001. Escherichia coli O157 infections and unpasteurised milk. Euro Surveill. 6:147–151.[Medline]

Barrett, N. J. 1986. Communicable disease associated with milk and dairy products in England and Wales: 1983–1984. J. Infect. 12:265–272.[Medline]

Bielaszewska, M., J. Janda, K. Blahova, H. Minarikova, E. Jikova, M. A. Karmali, J. Laubova, J. Sikulova, M. A. Preston, R. Khakhria, H. Karch, H. Klazarova, and O. Nyc. 1997. Human Escherichia coli O157:H7 infection associated with the consumption of unpasteurized goat’s milk. Epidemiol. Infect. 119:299–305.[Medline]

Blanco, M., J. E. Blanco, A. Mora, J. Rey, J. M. Alonso, M. Hermoso, J. Hermoso, M. P. Alonso, G. Dahbi, E. A. Gonzalez, M. I. Bernardez, and J. Blanco. 2003. Serotypes, virulence genes and intimin types of Shiga toxin-producing Escherichia coli isolated from healthy sheep in Spain. J. Clin. Microbiol. 41:1351–1356.[Abstract/Free Full Text]

Boerlin, P., S. A. McEwen, F. Boerlin-Petzold, J. B. Wilson, R. P. Johnson, and C. L. Gyles. 1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J. Clin. Microbiol. 37:497–503.[Abstract/Free Full Text]

Burnens, A. P., A. Frey, H. Lior, and J. Nicolet. 1995. Prevalence and clinical significance of verocytotoxin-producing Escherichia coli (VTEC) isolated from cattle in herds with and without calf diarrhoea. J. Vet. Med. B 42:311–318.

Chiodini, R. J., and J. Hermon-Taylor. 1993. The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteurization. J. Vet. Diagn. Invest. 5:629–631.[Free Full Text]

Cody, S. H., S. L. Abbott, A. A. Marfin, B. Schulz, P. Wagner, K. Robbins, J. C. Mohle-Boetani, and D. J. Vugia. 1999. Two outbreaks of multidrug-resistant Salmonella serotype Typhimurium DT104 infections linked to raw-milk cheese in Northern California. J. Am. Med. Assoc. 281:1805–1810.[Abstract/Free Full Text]

Corti, S., and R. Stephan. 2002. Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 26:15.

Cousins, D. V., R. Whittington, I. Marsh, A. Masters, R. J. Evans, and P. Kluver. 1999. Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable by IS900 polymerase chain reaction: Implications for diagnosis. Mol. Cell. Prob. 14:431–442.

De Buyser, M. L., B. Dufour, M. Maire, and V. Lafarge. 2001. Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. Int. J. Food Microbiol. 67:1–17.[Medline]

De Valk, H., E. Delarocque-Astagneaue, G. Colomb, S. Ple, E. Godard, V. Vaillant, S. Haeghebaert, P. H. Bouvet, F. Grimont, P. Grimot, and J. C. Desenclos. 2000. A community-wide outbreak of Salmonella enterica serotype Typhimurium infection associated with eating a raw milk soft cheese in France. Epidemiol. Infect. 124:1–7.[Medline]

Djonne, B., M. R. Jensen, I. R. Grant, and G. Holstad. 2003. Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway. Vet. Microbiol. 92:135–143.[Medline]

Englund, S., G. Bölske, and K. E. Johansson. 2002. An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol. Lett. 209:267–271.[Medline]

Foschino, R., A. Invernizzi, R. Barucco, and K. Stradiotto. 2002. Microbial composition, including the incidence of pathogens, of goat milk from the bergamo region of Italy during a lactation year. J. Dairy Res. 69:213–225.[Medline]

Grant, I. R., H. J. Ball, and M. T. Rowe. 1998. Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis. Lett. Appl. Microbiol. 26:166–170.[Medline]

Grant, I. R., E. I. Hitchings, A. McCarteny, F. Ferguson, and M. T. Rowe. 2002. Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk. Appl. Environ. Microbiol. 68:602–607.[Abstract/Free Full Text]

Grant I. R., L. M. O’Riordan, H. J. Ball, and M. T. Rowe. 2001. Incidence of Mycobacterium paratuberculosis in raw sheep and goats’ milk in England, Wales and Northern Ireland. Vet. Microbiol. 79:123–131.[Medline]

Guinée, P. A., W. H. Jansen, T. Wadström, and R. Sellwood. 1981. Escherichia coli associated with neonatal diarrhoea in piglets and calves. Curr. Top. Vet. Anim. Sci. 13:126–162.

Harrington, P., J. Archer, J. P. Davis, D. R. Croft, J. K. Varma, and EIC officers. 2002. Outbreak of Campylobacter jejuni infections associated with drinking unpasteurized milk procured through a cow-leasing program-Wisconsin, 2001. MMWR 51:548–549.[Medline]

Hermon-Taylor, J., T. J. Bull, J. M. Sheridan, J. Cheng, M. L. Stellakis, and N. Sumar. 2000. Causation of Crohn’s disease by Mycobacterium avium subspecies paratuberculosis. Can. J. Gastroenterol. 14:521–539.[Medline]

Hutchinson, D. N., F. J. Bolton, W. C. Jelley, W. G. Mathews, D. R. Telford, D. E. Counter, E. G. Jessop, and S. D. Horsley. 1985. Campylobacter enteritis associated with consumption of raw goat’s milk. Lancet 1:1037–1038.

Kalman, M., E. Szollosi, B. Czermann, M. Zimanyi, S. Szekeres, and M. Kalman. 2000. Milkborne campylobacter infection in Hungary. J. Food Prot. 63:1426–1429.[Medline]

Keene, W. E., K. Hedberg, D. E. Herriott, D. D. Hancock, R. W. McKay, T. J. Barrett, and D. W. Fleming. 1997. A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J. Infect. Dis. 176:815–818.[Medline]

Keswani, J., and J. F. Frank. 1998. Thermal inactivation of Mycobacterium paratuberculosis in milk. J. Food Prot. 61:974–978.[Medline]

Little, C. L., and J. De Louvois. 1999. Health risks associated with unpasteurized goats’ and ewes’ milk on retail sale in England and Wales. A PHLS Dairy Products Working Group Study. Epidemiol. Infect. 122:403–408.[Medline]

Martin, M. L., L. D. Shipman, J. G. Wells, M. E. Potter, K. Hedberg, I. K. Wachsmuth, R. V. Tauxe, J. P. Davies, J. Arnoldi, and J. Tilleli. 1986. Isolation of Escherichia coli O157:H7 from dairy cattle associated with two cases of hemolytic uremic syndrome. Lancet ll:1043.

McIntyre, L., J. Fung, A. Paccagnella, J. Isaac-Renton, F. Rockwell, B. Emerson, and T. Preston. 2002. Escherichia coli O157 outbreak associated with the ingestion of unpasteurized goat’s milk in British Columbia, 2001. Can. Commun. Dis. Rep. 28:6–8.[Medline]

Morgan, R., T. Massouras, M. Barbosa, L. Roseiro, F. Ravasco, I. Kandarakis, V. Bonnin, M. Fistakoris, E. Anifantakis, G. Jaubert, and D. Raynal-Ljutovac. 2003. Characteristics of goat milk collected from small and medium enterprises in Greece, Portugal and France. Small Rumin. Res. 47:39–49.

Pearce, L. E., H. Tuan Truong, R. A. Crawford, G. F. Yates, S. Cavaignac, and G. W. de Lisle. 2001. Effect of turbulent-flow pasteurisation on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk. Appl. Environ. Microbiol. 67:3964–3969.[Abstract/Free Full Text]

Piérard, D., G. Muyldermans, L. Moriau, D. Stevens, and S. Lauwers. 1998. Identification of new Verocytotoxin type 2 variant B-subunit genes in human and animal Escherichia coli isolates. J. Clin. Microbiol. 36:3317–3322.[Abstract/Free Full Text]

Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells, B. R. Davis, R. J. Herbert, E. S. Olcott, L. M. Johnson, N. T. Hargrett, P. A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681–685.[Abstract]

Roberts, D. 1985. Microbiological aspects of goat’s milk. A public health laboratory service survey. J. Hyg. 94:31–44.

Schmidt, H., L. Beutin, and H. Karch. 1995. Molecular analysis of the plasmid encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect. Immunol. 63:1055–1061.[Abstract]

Schmidt, H., H. Rüssmann, A. Schwarzkopf, S. Aleksic, J. Heesemann, and H. Karch. 1994. Prevalence of attaching and effacing Escherichia coli in stool samples from patients and controls. Zentbl. Bakteriol. 281:201–213.

Stephan, R., and K. Bühler. 2001. A survey of the prevalence of E. coli O157 and other Shigatoxin-producing E. coli in bulk-tank milk samples from north-east Switzerland. Arch. Lebensmittelhyg. 52:122–123.

Stephan, R., K. Bühler, and C. Lutz. 2002. Prevalence of genes encoding enterotoxins, exfoliative toxins and toxic shock syndrome toxin 1 in S. aureus strains isolated from bulk-tank milk samples in Switzerland. Milchwissenschaft 57:502–504.

Streeter, R. N., G. F. Hoffsis, S. Bech-Nielsen, W. P. Shulaw, and D. M. Rings. 1995. Isolation of Mycobacterium paratuberculosis from colostrum and milk of subclinically infected cows. Am. J. Vet. Res. 56:1322–1324.[Medline]

Sung, N., and M. T. Collins. 1998. Thermal tolerance of Mycobacterium paratuberculosis. Appl. Environ. Microbiol. 64:999–1005.[Abstract/Free Full Text]

Yamamoto, T., and M. Nakazawa. 1997. Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea. J. Clin. Microbiol. 35:223–227.[Abstract]

This article has been cited by other articles:

				R. Stephan, S. Schumacher, S. Corti, G. Krause, J. Danuser, and L. Beutin Prevalence and Characteristics of Shiga Toxin-Producing Escherichia coli in Swiss Raw Milk Cheeses Collected at Producer Level J Dairy Sci, July 1, 2008; 91(7): 2561 - 2565. [Abstract] [Full Text] [PDF]

				W. Y. Chen, M. H. Weng, S. E. Chen, H. C. Peh, W. B. Liu, T. C. Yu, M. C. Huang, M. T. Chen, H. Nagahata, and C. J. Chang Profile of Gelatinolytic Capacity of Raw Goat Milk and the Implications for Milk Quality J Dairy Sci, November 1, 2007; 90(11): 4954 - 4965. [Abstract] [Full Text] [PDF]

				C. Gonzalo, J. A. Carriedo, E. Beneitez, M. T. Juarez, L. F. De La Fuente, and F. San Primitivo Short Communication: Bulk Tank Total Bacterial Count in Dairy Sheep: Factors of Variation and Relationship with Somatic Cell Count J Dairy Sci, February 1, 2006; 89(2): 549 - 552. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Muehlherr, J. E. Articles by Stephan, R. Search for Related Content PubMed PubMed Citation Articles by Muehlherr, J. E. Articles by Stephan, R.