Survival curves of Listeria monocytogenes in chorizos modeled with artificial neural networks.

Using artificial neural networks (ANNs), a highly accurate model was developed to simulate survival curves of Listeria monocytogenes in chorizos as affected by the initial water activity (a(w0)) of the sausage formulation, temperature (T), and air inflow velocity (F) where the sausages are stored. The ANN-based survival model (R(2)=0.970) outperformed the regression-based cubic model (R(2)=0.851), and as such was used to derive other models (using regression) that allow prediction of the times needed to drop count by 1, 2, 3, and 4 logs (i.e., nD-values, n=1, 2, 3, 4). The nD-value regression models almost perfectly predicted the various times derived from a number of simulated survival curves exhibiting a wide variety of the operating conditions (R(2)=0.990-0.995). The nD-values were found to decrease with decreasing a(w0), and increasing T and F. The influence of a(w0) on nD-values seems to become more significant at some critical value of a(w0), below which the variation is negligible (0.93 for 1D-value, 0.90 for 2D-value, and <0.85 for 3D- and 4D-values). There is greater influence of storage T and F on 3D- and 4D-values than on 1D- and 2D-values.

Modeling the survival of Salmonella spp. in chorizos.

The survival of Salmonella spp. in chorizos has been studied under the effect of storage conditions; namely temperature (T=6, 25, 30 degrees C), air inflow velocity (F=0, 28.4 m/min), and initial water activity (a(w0)=0.85, 0.90, 0.93, 0.95, 0.97). The pH was held at 5.0. A total of 20 survival curves were experimentally obtained at various combinations of operating conditions. The chorizos were stored under four conditions: in the refrigerator (Ref: T=6 degrees C, F=0 m/min), at room temperature (RT: T=25 degrees C, F=0 m/min), in the hood (Hd: T=25 degrees C, F=28.4 m/min), and in the incubator (Inc: T=30 degrees C, F=0 m/min). Semi-logarithmic plots of counts vs. time revealed nonlinear trends for all the survival curves, indicating that the first-order kinetics model (exponential distribution function) was not suitable. The Weibull cumulative distribution function, for which the exponential function is only a special case, was selected and used to model the survival curves. The Weibull model was fitted to the 20 curves and the model parameters (alpha and beta) were determined. The fitted survival curves agreed with the experimental data with R(2)=0.951, 0.969, 0.908, and 0.871 for the Ref, RT, Hd, and Inc curves, respectively. Regression models relating alpha and beta to T, F, and a(w0) resulted in R(2) values of 0.975 for alpha and 0.988 for beta. The alpha and beta models can be used to generate a survival curve for Salmonella in chorizos for a given set of operating conditions. Additionally, alpha and beta can be used to determine the times needed to reduce the count by 1 or 2 logs t(1D) and t(2D). It is concluded that the Weibull cumulative distribution function offers a powerful model for describing microbial survival data. A comparison with the pathogen modeling program (PMP) revealed that the survival kinetics of Salmonella spp. in chorizos could not be adequately predicted using PMP which underestimated the t(1D) and t(2D). The mean of the Weibull probability density function correlated strongly with t(1D) and t(2D), and can serve as an alternative to the D-values normally used with first-order kinetic models. Parametric studies were conducted and sensitivity of survival to operating conditions was evaluated and discussed in the paper. The models derived herein provide a means for the development of a reliable risk assessment system for controlling Salmonella spp. in chorizos.

Reduction of Escherichia coli O157:H7 and Salmonella typhimurium in artificially contaminated alfalfa seeds and mung beans by fumigation with ammonia.

Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium. Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins. Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting. Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide. Samples were taken after intervals up to 22 h at 20 degrees C. Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans. Greater kills are apparently associated with lower initial bacterial loads. Germination of these seeds was unaffected by the treatment. It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.

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.

First findings of Cryptosporidium and Giardia in California sea lions (Zalophus californianus).

We report the detection and identification of Cryptosporidium and Giardia from 1 of 3 species of pinnipeds. Fecal samples were collected from Pacific harbor seal (Phoca vitulina richardsi), northern elephant seal (Mirounga angustirostris), and California sea lion (Zalophus californianus) in the northern California coastal area. By means of fluorescently labeled monoclonal antibodies, Cryptosporidium oocysts were detected in 3 samples from California sea lions, 1 of which also contained Giardia cysts. Oocysts of Cryptosporidium and cysts of Giardia were morphologically indistinguishable from oocysts of C. parvum and cysts of G. duodenalis from other animal origins. Oocysts and cysts were then purified using immunomagnetic separation techniques and identified by polymerase chain reaction (PCR), from which species-specific products were obtained. Sequence analysis revealed that the 452-bp and 358-bp PCR products of Cryptosporidium isolated from California sea lion had identities of 98% with sequences of their template fragments of C. parvum obtained from infected calves. Based on morphological, immunological, and genetic characterization, the isolates were identified as C. parvum and G. duodenalis, respectively. The findings suggested that California sea lions could serve as reservoirs in the environmental transmission of Cryptosporidium and Giardia.

Comparative detection of Cryptosporidium parvum oocysts from apple juice.

Drinking unpasteurized apple juice (or cider) has been associated with cryptosporidiosis, the diarrheal disease caused by the small protozoan parasite, Cryptosporidium parvum. This report compares detection of C. parvum oocysts from apple juice by acid-fast staining (AFS), direct immunofluorescence assay (DIFA), and polymerase chain reaction (PCR), following sample concentration by formalin-ethyl acetate sedimentation or sucrose flotation. Flotation was more efficient than sedimentation in recovering oocysts, and DIFA consistently detected lower numbers of oocysts than AFS. In combination, flotation-AFS could detect 3000 to 10,000 oocysts inoculated into 100 ml of apple juice while flotation-DIFA was able to detect as few as 100 oocysts. The highest sensitivity, 10 to 30 oocysts per 100 ml of apple juice, was achieved by DIFA following immunomagnetic capture (IC) of oocysts from samples concentrated by the flotation method. The detection limit of PCR following flotation or flotation IC was 30 to 100 oocysts; sequence analysis of the amplicon demonstrated that the PCR amplicon was C. parvum-specific.

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns.

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.

Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures.

Exponential inactivation was observed for Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure with decimal reduction times ranging from half a day at 37 C to 1-2 wk at 4 C. There was no material difference in inactivation rates between S. typhimurium and E. coli O157:H7. Inactivation was slower in slurries made by mixing two parts of water with one part of manure; decimal reduction times (time required for 90% destruction) ranged from 1-2 days at 37 C to 6-22 wk at 4 C. Escherichia coli O157:H7 consistently exhibited slightly slower inactivation than S. typhimurium. Log decimal reduction time for both strains was a linear function of storage temperature for manure and slurries. Chemical analysis indicated that accumulation of free ammonia in poultry manure was an important factor in inactivation of the pathogens. This finding was experimentally confirmed for S. typhimurium by adding ammonia directly to peptone water or to bovine manure, which was naturally low in ammonia, and adjusting pH to achieve predetermined levels of free ammonia.

Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals.

We tested an improved immunofluorescence assay (IFA) in detecting Giardia and Cryptosporidium from feces of asymptomatic adult cervine animals. Samples were concentrated by sucrose flotation before being stained by fluorescent monoclonal antibody and examined microscopically. The detection limit was determined as 500 G. intestinalis cysts or 200 C. parvum oocysts/g of sample. Among the 82 samples collected from adult fallow deer, Columbian black-tailed deer, and Tule elk in northern California, 3 (3.7%) contained G. intestinalis cysts, which were confirmed by a species-specific polymerase chain reaction (PCR) following immunomagnetic capture (IC) of cysts. C. parvum oocysts were detected in a total of 13 (15.9%) samples, and oocysts from 2 such samples were smaller than oocysts from the other 11 samples. C. parvum identification was also confirmed by specific IC-PCR and sequencing of the PCR product. In addition, a C. muris-like organism was detected in 2 (2.4%) samples. Findings obtained with the improved IFA confirmed that cysts/oocysts may pass unnoticed in adult cervine animals and that subclinically infected individuals could serve as potential carriers of infection for humans and other animals via contaminated feces or water.

Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

Cryptosporidium parvum studies with dairy products.

Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice-cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation-based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air-drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at -20 degrees C. Although approximately 20% (25 and 18%) of oocysts were viable before hardening, none were viable after 24 h at -20 degrees C. Control samples of oocysts suspended in distilled water and stored at -20 degrees C were taken at the same time intervals and 8% of the oocysts were still viable after 24 h.

Removal of Escherichia coli O157:H7 from surface tissues of beef carcasses inoculated with wet and dry manure.

Beef tissues were contaminated with wet and dry manure. The manure was previously inoculated with Escherichia coli O157:H7 GFP, genetically modified with a plasmid encoding a protein that fluoresces green when exposed to long-wave ultraviolet light. After incubation at 37 degrees C for 5 days, the wet manure was spread on the surface of beef tissues at an average E. coli O157:H7 GFP level of 6.62 log CFU/cm2. Dry manure was obtained by subjecting wet manure to natural drying (simulating dry manure adhering to the hides of cattle) and was also applied to the surfaces of beef tissues. The degree of removal of E. coli O157:H7 GFP by washing was compared to the removal of cells of the same strain that had been inoculated as a suspension. The E. coli O157:H7 mixed into feces of cattle adhered more strongly to meat surfaces than that applied as a suspension, complicating the removal by conventional washing procedures. The fate of the bacterium mixed into wet or dry manure was evaluated. An initial decrease of the inoculated population was observed; this was probably an effect of the changed environment represented by the manure. After adaptation, the inoculated bacteria grew in the wet manure; a maximum population was reached in 5 days at 37 degrees C; levels declined with drying. The use of the GFP marker was of great value, since it allowed enumeration of E. coli O157:H7 in the presence of the natural flora of manure.

Decontaminating beef for Escherichia coli O157:H7.

Beef lean, fat, and connective tissues were inoculated with Escherichia coli O157:H7 before and after a prewashing procedure to compare the efficacy of prewashing and no prewashing on bacterial adherence and, consequently, on the removal of bacteria from the inoculated surfaces. Prewashing consisted of spraying tissues with tap water before inoculation. Final washing with disinfectant solutions compared the efficacy of several chemicals for the removal or destruction of E. coli O157:H7. The results showed that prewashing was very effective in reducing the numbers of bacterial cells on beef tissues, mainly lean tissue, in the control samples which received final washing with water. An opposite effect of prewashing was observed when disinfectant solutions were used for final washing; this may be due to dilution by water carried on the tissues after prewashing. The efficacy of chemicals was dependent on the type of exposed tissue. Hydrogen peroxide (3%) was more efficient in the removal of E. coli O157:H7 from connective tissues, with reductions greater than 4 log CFU/cm2, compared to a normally washed control (P < 0.01). Chlorhexidine (0.1%) was very efficient on fat and lean tissues, causing reductions over 5 log CFU/cm2 on not prewashed fat and lean tissues, compared to the control (P < 0.01). Acetic acid (5%) was the least effective, decreasing the number of CFU by under 1 log/cm2 as compared to the control; and no statistically significant difference was found among tissues, even though the removal of bacteria seemed less in lean tissue compared to fat or connective tissues.

Differentiation of Cryptosporidium parvum isolates by a simplified randomly amplified polymorphic DNA technique.

Genomic DNA was isolated from Cryptosporidium parvum oocysts by a specific immunomagnetic separation-in vitro excystation procedure and subjected to randomly amplified polymorphic DNA analysis using sequence-independent primers. An estuary C. parvum isolate was easily differentiated from several bovine isolates, while five bovine isolates of the same origin were indistinguishable from each other.

Microbial food borne pathogens. Escherichia coli O157:H7.

Escherichia coli O157:H7 has emerged in the past 20 years as a pathogen of public health importance. Although most E. coli are normal flora in the colons of humans and other warm-blooded animals, several strains are capable of causing disease in humans. In recent years, E. coli O157:H7 and other shiga-like toxin-producing strains have been transmitted via foods and caused disease ranging from bloody diarrhea, and in more severe cases, hemolytic uremic syndrome and thrombocytopenic purpura. The reservoir appears to be cattle and, perhaps, other ruminants. Control is difficult in nonheated foods due to the organism's tolerance to low pH. Only supportive, symptomatic treatment is available for affected humans, and means to eliminate carriage in livestock are not presently available.

Cryptosporidium parvum development in the BS-C-1 cell line.

Cryptosporidium parvum is a worldwide parasitic protozoon capable of causing life-threatening disease in immunocompromised patients. In vitro cultivation of C. parvum has been under investigation for development of a well-defined in vitro model for C. parvum infectivity assay. This is the first report of C. parvum completing its life cycle in BS-C-1, an African green monkey kidney cell line. Both sodium hypochlorite-stimulated oocysts and purified sporozoites were able to initiate infection that led to completion of the whole life cycle, although inoculating purified sporozoites was less efficient. Beside Giemsa staining and normal light microscopy, an indirect fluorescent antibody staining method was developed to facilitate the detection and interpretation of C. parvum life stages developed in vitro. A C. parvum-specific polymerase chain reaction was also applied to detect and confirm the presence of C. parvum life stages in liquid from oocyst-infected cell cultures. In addition to the 452-base-pair (bp) product that can be specifically amplified from C. parvum oocysts and sporozoites, a fragment near 280 bp was also obtained. Various applications of this in vitro culture system are envisioned.