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Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil.

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk - types B and C - of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1%) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

Animals↗

Therapy of Venezuelan patients with severe mucocutaneous or early lesions of diffuse cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus Calmette-Guerin: preliminary report.

Severe mucocutaneous (MCL) and diffuse (DCL) forms of American cutaneous leishmaniasis (ACL) are infrequent in Venezuela. Chemotherapy produces only transitory remission in DCL, and occasional treatment failures are observed in MCL. We have evaluated therapy with an experimental vaccine in patients with severe leishmaniasis. Four patients with MCL and 3 with early DCL were treated with monthly intradermal injections of a vaccine containing promastigotes of Leishmania (Viannia) braziliensis killed by pasteurization and viable Bacillus Calmette- Guerin. Clinical and immunological responses were evaluated. Integrity of protein constituents in extracts of pasteurized promastigotes was evaluated by gel electrophoresis. Complete remission of lesions occurred after 5-9 injections in patients with MCL or 7-10 injections in patients with early DCL. DCL patients developed positive skin reactions, average size 18.7 mm. All have been free of active lesions for at least 10 months. Adverse effects of the vaccine were limited to local reactivity to BCG at the injection sites and fever in 2 patients. Extracts of pasteurized and fresh promastigotes did not reveal differences in the integrity of protein components detectable by gel electrophoresis. Immunotherapy with this modified vaccine offers an effective, safe option for the treatment of patients who do not respond to immunotherapy with vaccine containing autoclaved parasites or to chemotherapy.

Adult↗

[Effect of in-home boiling and refrigeration on bacterial load of pasteurized milk]

OBJECTIVES: To assess the efficacy of in-home boiling of pasteurized milk in reducing the bacterial load and the ability of the refrigeration in preserving the milk after boiling.METHODS: Thirty samples of pasteurized milk bought in São Paulo, Brazil, were submitted to in-home boiling procedure at the laboratory. Portions of samples were taken before and after boiling, and after 2, 4, 6 and 24 hours under refrigeration for microbiological analyses. Methods used were mesophilic bacteria count, and coliforms and Escherichia coli (E.coli) enumeration.RESULTS: No sample presented mesophilic bacteria count above the Brazilian standard for pasteurized milk. E.coli was not recovered from any sample. Ten samples (33%) had coliform bacteria; of these, 3 samples (10%) were above the standard. Mesophilic bacteria count after boiling was significantly lower than before boiling. After 24 hours under refrigeration, mesophilic bacteria count was significantly higher than after boiling. No significant differences were found between the intervals of 2, 4, 6 and 24 hours under refrigeration. Samples before boiling presented significantly higher coliform bacteria. No coliform bacteria were recovered at any time after boiling.CONCLUSIONS: In-home boiling of milk reduced bacterial load, while refrigeration kept bacteria under low counts.

Journal Article↗

Pasteurization of discard mycoplasma mastitic milk used to feed calves: thermal effects on various mycoplasma.

Discard milk from sick or antibiotic-treated cows is often used as an economical alternative to milk replacer at dairy farms. This practice poses a health risk to calves if the discard milk is from cows with mycoplasma mastitis. Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma canadense are among the agents known to cause contagious mastitis in cattle and occasionally pneumonia, otitis media, or arthritis in calves. This report describes a recent outbreak of calf polyarthritis and respiratory disease on a midwest dairy farm. The farm fed discard mycoplasma mastitic milk to its calves. On-the-farm pasteurization of the discard milk to 65 degrees C for 1 h before feeding prevented additional illness in the calves. Discard milk samples were collected before and after heating and tested for mycoplasma by culture. Only samples collected before pasteurization yielded live cultures. Common mastitic mycoplasma agents were also tested for sensitivity to heat. It was determined that 65 degrees C killed M. bovis and M. californicum after 2 min of exposure, while M. canadense remained viable for up to 10 min. Exposure to 70 degrees C inactivated M. bovis and M. californicum after 1 min, but M. canadense samples were positive for up to 3 min. Thus, M. canadense appears to be more heat resistant than M. bovis and M. californicum. Heat treatment that results in the destruction of M. canadense should be used for the pasteurization of discard mycoplasma mastitic milk.

Animal Feed↗

The survival of foot-and-mouth disease virus in raw and pasteurized milk and milk products.

The Foot-and-Mouth Disease virus (FMDV) is not a public health threat, but it is highly contagious to cloven-footed animals. The virus is shed into milk up to 33 h before there are apparent signs of the disease in dairy cows, and, in extreme cases, signs of disease may not appear for up to 14 d. During this time, raw milk can serve as a vector for spread of the disease both at the farm and during transport to the processing plant by milk tanker. Raw milk and milk products fed to animals have the potential to cause infection, but the potential for pasteurized milk products to cause infection is largely unknown. Current minimum pasteurization standards may not be adequate to eliminate FMDV in milk completely. The purpose of this paper is to assess the literature on the thermal resistance of FMDV in milk and milk products, to identify the risks associated with ingestion of pasteurized products by animals, and to lay a strategy to prevent the spread of FMDV from contaminated milk.

Animals↗

Effect of vacuum-condensed or ultrafiltered milk on pasteurized process cheese.

Milk was concentrated by ultrafiltration (UF) or vacuum condensing (CM) and milks with 2 levels of protein: 4.5% (UF1 and CM1) and 6.0% (UF2 and CM2) for concentrates and a control with 3.2% protein were used for manufacturing 6 replicates of Cheddar cheese. For manufacturing pasteurized process cheese, a 1:1 blend of shredded 18- and 30-wk Cheddar cheese, butter oil, and disodium phosphate (3%) was heated and pasteurized at 74 degrees C for 2 min with direct steam injection. The moisture content of the resulting process cheeses was 39.4 (control), 39.3 (UF1), 39.4 (UF2), 39.4 (CM1), and 40.2% (CM2). Fat and protein contents were influenced by level and method of concentration of cheese milk. Fat content was the highest in control (35.0%) and the lowest in UF2 (31.6%), whereas protein content was the lowest in control (19.6%) and the highest in UF2 (22.46%). Ash content increased with increase in level of concentration of cheese milk with no effect of method of concentration. Meltability of process cheeses decreased with increase in level of concentration and was higher in control than in the cheeses made with concentrated milk. Hardness was highest in UF cheeses (8.45 and 9.90 kg for UF1 and UF2) followed by CM cheeses (6.27 and 9.13 kg, for CM1 and CM2) and controls (3.94 kg). Apparent viscosity of molten cheese at 80 degrees C was higher in the 6.0% protein treatments (1043 and 1208 cp, UF2 and CM2) than in 4.5% protein treatments (855 and 867 cp, UF1 and CM1) and in control (557 cp). Free oil in process cheeses was influenced by both level and method of concentration with control (14.3%) being the lowest and CM2 (18.9%) the highest. Overall flavor, body and texture, and acceptability were higher for process cheeses made with the concentrates compared with control. This study demonstrated that the application of concentrated milks (UF or CM) for Cheddar cheese making has an impact on pasteurized process cheese characteristics.

Animals↗

Automated control and monitoring of thermal processing using high temperature, short time pasteurization.

High temperature, short time pasteurization was used to evaluate a computer-based system for controlling the pasteurization process, acquiring data, and monitoring records. Software was used for the control of hot water temperature, flow rate through the centrifugal timing pump, and diversion of under-processed product. Three types of control strategies were conducted: single loop, cascade, and multivariable. The single loop control strategy showed the most rapid responses to temperature changes, but the temperature response curve was slowest to return to its set point. The cascade control strategy showed slower recoveries to temperature changes, but the temperature response curve was smoother. The multivariable control strategy responded slightly faster than the cascade control strategy, and the temperature response curve was slightly smoother than the cascade control strategy. The multivariable control strategy was able to control the flow diversion valve by the use of a lethality controller. The data acquisition system, used to monitor the data obtained from the high temperature, short-time pasteurization system, was within +/- 0.1 degree C of the temperature recorded by the safety thermal limit recorder. Reliability was determined by examining the changes in the position of the flow diversion valve to identify process deviations and by comparing the changes to the event marker on circular charts. The data acquisition system was an effective alternative for monitoring the completeness of data.

Animals↗

Gamma-glutamyltransferase as a marker for the pasteurization of raw milk.

The degree and rate of inactivation of gamma-glutamyltransferase in raw cow's milk by heating at 50, 60, 70, and 80 degrees C for 1, 2, 3, 5, 10, 15, 20, 25, and 30 min were measured to evaluate the suitability of this enzyme as a marker for the pasteurization of milk. The enzymes alkaline phosphatase and lactate dehydrogenase were also measured under similar conditions for comparison. The patterns of heat inactivation of gamma-glutamyltransferase and alkaline phosphatase were similar, with only a minimal inactivation of the enzymes at 50 degrees C. The rate of inactivation increased as a result of increasing temperatures and time. A complete inactivation of both enzymes was seen at 70 degrees C after 10 min and at 80 degrees C after 1 min. Lactate dehydrogenase showed a higher heat resistance with almost complete inactivation at 70 degrees C for 30 min, and compete inactivation at 80 degrees C for 3 min. No activities of these enzymes were found in commercially pasteurized or heat-treated milk. The levels of gamma-glutamyltransferase in raw milk were between 8 and 10% higher than those of alkaline phosphatase and lactate dehydrogenase, making it more sensitive and accurate as a testing marker. It seems that gamma-glutamyltransferase may serve as a good pasteurization marker. Furthermore, the simplicity of testing and the availability of commercial kits for testing by both wet and dry chemistry make it an attractive choice, especially because dry chemistry procedures overcome the difficulties originating from the turbidity of milk, which interferes with spectrophotometric procedures.

Alkaline Phosphatase↗

Rapid hot dog surface pasteurization using cycles of vacuum and steam to kill Listeria innocua.

The vacuum/steam/vacuum surface pasteurization process was applied to hot dogs inoculated on the surface with non-pathogenic Listeria innocua. Using the optimum conditions previously found for processing chicken carcasses as a starting point, optimum process conditions were determined for a hot dog treatment compatible with current process line speed. Cycling the treatment significantly improved the microbiological kill. At the optimum conditions of steam time of 0.3 s at 138 degrees C (two cycles), a bacteria kill >3 log CFU/ml was attained. Pasteurization, frequently considered to be a kill of >5 log CFU/ml, was reached by increasing the number of cycles to three. The surface pasteurization process should ensure that hot dogs reaching the consumer are free of Listeria.

Animals↗

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

Adaptation, Physiological↗

Inactivation of Cryptosporidium parvum oocysts in cider by flash pasteurization.

Cryptosporidium parvum is a well-recognized pathogen of significant medical importance, and cider (apple juice) has been associated with foodborne cryptosporidiosis. This study investigated the effect of flash pasteurization on the viability of contaminant C. parvum oocysts. Cider inoculated with oocysts was heated at 70 or 71.7 degrees C for 5, 10, or 20 s, and oocyst viability was measured by a semiquantitative in vitro infectivity assay. By infecting multiple wells of confluent Madin-Darby bovine kidney cells with serial dilutions of heat-treated oocysts and examining infected cells by indirect fluorescent antibody staining, the most probable number technique was applied to quantify log reduction of oocyst viability. Heating for 10 or 20 s at either temperature caused oocyst killing of at least 4.9 log (or 99.999%), whereas oocyst inactivation after pasteurization for 5 s at 70 and 71.7 degrees C was 3.0 log (99.9%) and 4.8 log (99.998%), respectively. Our results suggested that current practices of flash pasteurization in the juice industry are sufficient in inactivating contaminant oocysts.

Animals↗

Use of pulsed ultraviolet laser light for the cold pasteurization of bovine milk.

Because of concerns that some potentially dangerous microorganisms may survive conventional heat pasteurization of milk and because the heat needed to sterilize milk affects marketability, the ability to efficiently cold pasteurized milk may become more desirable. In this pilot study, we investigated the use of pulsed ultraviolet (PUV) laser light to nonthermally (cold) pasteurized bovine milk. Dairy bulk tank milk was treated with UV light (248 nm) emitted from a pulsed excimer laser. The samples were then analyzed for surviving bacteria by spiral plate counting and subculturing in Trypticase soy broth. Other bulk tank milk samples were inoculated with one of eight relevant milk bacterial species before being exposed to laser light. There was no growth observed for any of the plated or subcultured samples exposed to 25 J/cm2. One bacterial isolate was then used to inoculate milk to further investigate bactericidal laser light doses. Growth was observed for samples treated with an average of 0.3 to 6.6 J/cm2 but not for those treated with 12.6 J/cm2. The results indicate that in principle, the bacterial content of milk can be adequately controlled by exposure to PUV laser light.

Animals↗

Inhibition of Listeria monocytogenes in pH-adjusted pasteurized liquid whole egg.

Although the transmission of L. monocytogenes to humans via pasteurized egg products has not been documented, L. monocytogenes and other Listeria species have been isolated from commercially broken raw liquid whole egg (LWE) in both the United States and Ireland. Recent Listeria thermal inactivation studies indicate that conventional minimal egg pasteurization processes would effect only a 2.1- to 2.7-order-of-magnitude inactivation of L. monocytogenes in LWE; thus, the margin of safety provided by conventional pasteurization processes is substantially smaller for L. monocytogenes than for Salmonella species (a 9-order-of-magnitude process). The objective of this study was to evaluate the inhibitory effects of nisin on the survival and growth of L. monocytogenes in refrigerated and pH-adjusted (pH 6.6 versus pH 7.5) ultrapasteurized LWE and in a liquid model system. The addition of nisin (1,000 IU/ml) to pH-adjusted ultrapasteurized LWE reduced L. monocytogenes populations by 1.6 to > 3.3 log CFU/ml and delayed (pH 7.5) or prevented (pH 6.6) the growth of the pathogen for 8 to 12 weeks at 4 and 10 degrees C. Bioactive nisin was detected in LWE at both pH values for 12 weeks at 4 degrees C. In subsequent experiments, Listeria reductions of > 3.0 log CFU/ml were achieved within 24 h in both LWE and broth plus nisin (500 IU/ml) at pH 6.6 but not at pH 7.5, and antilisterial activity was enhanced when nisin was added as a solution rather than in dry form.

Anti-Bacterial Agents↗

Computational fluid dynamics analysis for process impact assessment during thermal pasteurization of intact eggs.

Transient temperature and albumen velocity profiles during thermal pasteurization of intact eggs were studied using a commercial computational fluid dynamics (CFD) package. Simulated temperature profiles were in close agreement with experimental data for eggs of different sizes. Convective heat transfer only occurred in the egg white fraction, and conductive heat transfer only occurred in the yolk. For process assessment, a generally accepted kinetic inactivation model for Salmonella Enteritidis was incorporated into the CFD analysis. Minimum process times and temperatures needed to provide equivalent pasteurization at 5-log reductions of the target microorganism were obtained on a theoretical basis. The combination of CFD analysis and inactivation kinetics can be very useful for assessing pasteurization of intact eggs and can enable processors to gain a better understanding of these processes and to establish process conditions for consumer-safe eggs.

Animals↗

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores.

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.

Animals↗

Comprehensive survey of pasteurized fluid milk produced in the United States reveals a low prevalence of Listeria monocytogenes.

A comprehensive survey was undertaken to generate contemporary data on the prevalence of Listeria monocytogenes in pasteurized fluid milk produced in the United States. Samples (5,519) near the sell-by expiration date were purchased at retail outlets over a 5-week period and analyzed for presence of L. monocytogenes. Products consisted of whole milk, nonfat milk, and chocolate milk packaged in gallon, half gallon, quart, pint, and half-pint containers. Samples were collected from both large and small retail stores in urban and suburban locations in four FoodNet cities (Baltimore, Md., Atlanta, Ga., St. Paul/ Minneapolis, Minn., and San Francisco, Calif.). Samples were prescreened for L. monocytogenes by the AOAC-approved rapid Vitek immunodiagnostic assay system, enzyme-linked fluorescent assay method. Positive prescreening samples were cultured according to the Bacteriological Analytical Manual, enumerated for L. monocytogenes with a nine-tube most-probable-number (MPN) procedure, and confirmed by biochemical characterization. The frequency of isolation of L. monocytogenes in these products was 0% (0 of 1,897) in whole milk, 0.05% (1 of 1,846) in nonfat milk, 0% (0 of 1,669) in chocolate milk, and 0% (0 of 107) in other (reduced fat and low fat) milk samples. Overall, L. monocytogenes was confirmed in only 0.018% of pasteurized milk samples (1 of 5,519). Enumeration of the single confirmed positive nonfat milk sample revealed low-level contamination (<0.3 MPN/g), even when sampled 5 days past the expiration of the sell-by date. The results confirm the low frequency of contamination of pasteurized fluid milk products by L. monocytogenes for products sold in the United States and reaffirm the reduction of contamination frequency of fluid milk by L. monocytogenes when compared with earlier estimates from the U.S. Food and Drug Administration Dairy Safety Initiatives Program.

Animals↗

[Intravesical BCG-therapy: comparison of side effects of Connaught (Toronto) and Pasteur (Paris) strains].

INTRODUCTION: Urologists have felt that the adverse effects of intravesical BCG-therapy have been more serious and more frequent since the use of the Connaught strain. The objective of this retrospective study was to compare the toxicity of this new strain with that previously used in France (Pasteur strain). MATERIAL AND METHODS: After endoscopic resection, 89 patients with stage Ta grade 1-2 recurrent or T1 grade 3 and/or CIS bladder tumour were treated with 6 instillations of 150 mg of BCG Pasteur from 1992 to 1996 (50 patients: group 1) or 81 mg of BCG Connaught from January 1997 to December 1998 (39 patients: group 2). Adverse effects were classified as minor, lasting less than 48 hours (bladder irritation syndrome and/or macroscopic haematuria and/or fever less than 38 degrees C), moderate (requiring symptomatic treatment, reduction of the dose or an increased interval between instillations), and major (contraindication to continuation of treatment). RESULTS: 74% of patients in group 1 presented at least one adverse effect versus 77% in group 2. The reasons for permanent discontinuation of BCG-therapy in groups 1 and 2, respectively, were as follows: malaise during instillation (1 vs 0), bladder irritation syndrome not controlled by symptomatic treatment (4 vs 5) and epididymitis (0 vs 1). Pulmonary tuberculosis was diagnosed in one patient from group 2, one year after the last instillation. The frequency and severity of adverse effects were not statistically different between the two groups. The number of patients discontinuing BCG-therapy because of severe complications was also not statistically different between the two groups. CONCLUSION: This study did not reveal any difference of toxicity between Connaught and Pasteur strain in intravesical BCG-therapy of superficial bladder tumours.

Adjuvants, Immunologic↗

The heuristic function of 'error' in the scientific methodology of Louis Pasteur: the case of the silkworm diseases.

With the aid of the Cahiers de laboratoire, the Correspondence and, of course, the Oeuvre de Pasteur, this work reconstructs the extraordinary scientific undertakings of the great French scientist in his study of silkworm diseases. The focus of this study consists in the attempt to explain the initial perplexing behaviour of Pasteur, even in the presence of correct interpretations regarding the causes of these diseases (cfr. the results obtained by Béchamp); for a good three years he insisted on maintaining that the aetiology of silkworm diseases could not be attributed to pathogenetic germs from outside. And this was in spite of the fact that previously (through fermentation and spontaneous generation) he had been able to demonstrate the importance of microorganisms in biological processes. Finally it is intended to highlight the extraordinary methodological depth of that initial 'error', which was capable of paving the way for the future conquests of Pasteur in the field of aetiology and the prevention of infectious diseases.

Animal Diseases↗