Inactivation kinetics of a surrogate yield conservative predictions of foodborne pathogen reductions from low water activity foods of varying size and composition during low-temperature steam processing.

There is a growing interest in using models to predict foodborne pathogen inactivation as a way to validate or verify preventive controls. Unlike liquid foods, solid, low water activity foods (LWAF) are heterogenous in composition and structure and do not transfer heat uniformly. Using models constructed from one food to predict pathogen inactivation on another LWAF is complex and may not always be possible, even if the foods have similar composition. Using models constructed from inactivation kinetics of three foodborne pathogens and a surrogate from vacuum-steam-pasteurized (72 and 82 °C) whole macadamia nuts and dried apricot halves, 3-log reductions were predicted for the same pathogens and foods of reduced size. Model fits (First-order, Weibull, and Gompertz) were significantly impacted by the food type regardless of particle size. Despite the foods being identical in composition with particle size as the only altered characteristic, best-fit models accurately predicted the 3-log reductions only 50% of the time, but the surrogate inactivation models provided conservative predictions for pathogen reductions, highlighting that a surrogate's model may be a suitable tool for predicting pathogen reduction on LWAFs.

Effects of dirty housing and a Typhimurium DT104 challenge on pig growth performance, diet utilization efficiency, and gas emissions from stored manure.

The objectives of this study were to elucidate the effects of a dirty environment and a challenge plus associated environmental contamination on pig growth performance, diet utilization efficiency, and gas emissions (CO, NH, CH, NO, and HS) from stored manure. Twenty-four weaned barrows, aged 31 d at initiation of the trial, were randomly allotted to 3 different treatments in a completely randomized design. Treatments were: pigs housed in cages with manure removed and cages washed daily (Clean); pigs housed in cages sprayed daily with manure slurry mixtures (Dirty); or pigs challenged with Typhimurium DT104 and housed in cages that were not washed, but manure was removed daily ( challenge). Rectal temperature, body weight, daily feed intake, manure output, manure composition, and gas emissions from stored manure were measured throughout the 24-d animal phase. The Dirty and challenge treatments were statistically compared to the Clean treatment to evaluate individual effects. Dirty housing tended to decrease ADG from d 1 to 24 ( = 0.06) but there were no other effects on pig performance compared with the Clean treatment. In contrast, a challenge was associated with a marked reduction in each of the measured indicators of pig performance. challenge increased the carbon to nitrogen ratio, ether extract, and lignin concentrations in excreted manure ( = 0.02, 0.01, 0.003, respectively), and increased manure and head space temperatures in manure tanks ( < 0.0001). Gas emissions from stored manure of pigs on the Dirty or treatments were increased for each of the measured gases as compared to the Clean treatment ( < 0.01) when expressed per unit of BW gain. When gas emissions from manure of pigs housed in the Dirty treatment were expressed per unit of manure volatile solids (VS), they were increased for NH, CH, and HS ( < 0.02). challenge was associated with increased emissions of CO, and NO and decreased emissions of HS per kilogram manure VS compared to the Clean treatment ( = 0.06, 0.03, 0.04, respectively). Collectively, these results indicated that a challenge and associated housing contamination caused depressed growth rate and increased manure gas emissions, while exposure to a Dirty environment slightly reduced growth performance and clearly increased manure gas emissions per unit of BW gain as compared to Clean control.

Effects of porcine reproductive and respiratory syndrome virus on pig growth, diet utilization efficiency, and gas release from stored manure.

The objectives of this study were to examine the effects of porcine reproductive and respiratory syndrome virus (PRRSV) infection and vaccination on pig growth, dietary nutrient efficiency of utilization, manure output, and emissions of CO, CH, HS, NO, and NH gases from stored manure. Forty-eight pigs, aged 21 d at the start of the study, were subjected to 1 of 4 treatment combinations arranged in a 2 × 2 factorial design with main factors of PRRSV vaccination and PRRSV infection. Body weight, ADFI, manure output, and nutrient efficiency of utilization were assessed and gas emissions from stored manure were determined daily from 50 to 78 d of age and for 24 d after completion of the animal phase. Infection with PRRSV markedly reduced final BW, ADG, and ADFI ( < 0.01) and reduced efficiencies of ADF and ether extract utilization ( = 0.05 and = 0.02, respectively) regardless of vaccination status. No significant treatment effects were found on manure output, manure pH, efficiencies of lignin utilization, and N retention. Infecting pigs with PRRSV increased daily manure CO emission per pig ( = 0.01). There was an interaction between immunization and infection for NO per pig with manure from uninfected, vaccinated pigs producing as much as the manure from infected, vaccinated pigs whereas there was a difference by PRRSV infection state for nonvaccinated pigs. There were also interactions between treatments for HS and NO emissions per kilogram of manure volatile solids excreted ( = 0.01 and = 0.0001, respectively) with the same pattern as for NO per pig; that is, the vaccinated pigs had similar rates of emission regardless of infection state. Pigs infected with PRRSV increased NO nitrogen per kilogram of total N excreted compared with noninfected groups ( = 0.03). Collectively, these results indicated that PRRSV infection caused decreased growth rates and nutrient utilization efficiency and increased gas emissions from stored manure.

Postharvest transfer and survival of Salmonella enterica serotype enteritidis on living lettuce.

UNLABELLED: The potential for postharvest transfer of Salmonella to 'living lettuce' is not well understood. In this study, the transfer of Salmonella enterica Enteritidis (6 log CFU g(-1) ) from worker hands or contaminated roots to leaves of living lettuce was quantified. Transfer rates of Salmonella from contaminated gloves to sequentially handled lettuce heads ranged from 94% to head 1, 82% to head 2 and 69% to head 3. On average, 2.9 ± 0.1 log CFU g(-1) (64%) Salmonella was transferred from inoculated roots to leaves resulting from typical postharvest handling activities for living lettuce. Salmonella persisted on leaves stored at recommended storage temperatures (4°C) and increased 0.5 log CFU g(-1) when stored at temperature abuse conditions (12°C). Salmonella increased 1.6 log CFU g(-1) on roots after 18-day storage at 12°C, emphasizing the need to maintain temperature control to reduce the risk of human illness. SIGNIFICANCE AND IMPACT OF THE STUDY: Hydroponically grown lettuce packaged in plastic clamshells with intact roots, marketed as 'living lettuce', is increasing in popularity due to its extended shelf life. This study demonstrates the transfer of Salmonella from contaminated worker hands and contaminated roots to leaves where it persisted at 4°C for 18 day. Temperature abuse (12°C) increased Salmonella on roots and leaves. These findings suggest that failure to maintain temperatures below 12°C can pose a risk for consumers purchasing living lettuce at markets where recommended storage temperatures are not maintained.

Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons.

AIMS: To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. METHODS AND RESULTS: Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready-to-eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10°C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to γ-Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4°C but not at 10°C storage, thus highlighting the importance of temperature in fresh packaged spinach. CONCLUSIONS: The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture- and nonculture-dependent approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture-based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.

Use of Saccharomyces cerevisiae fermentation product on growth performance and microbiota of weaned pigs during Salmonella infection.

Anaerobically fermented yeast products are a rich source of nutritional metabolites, mannanoligosaccharides, and ß-glucans that may optimize gut health and immunity, which can translate into better growth performance and a reduced risk of foodborne pathogens. The objective of this study was to quantify the effects of Saccharomyces cerevisiae fermentation product (Diamond V Original XPC) inclusion in nursery diets on pig performance and gastrointestinal microbial ecology before, during, and after an oral challenge with Salmonella. Pigs (n = 40) were weaned at 21 d of age, blocked by BW, and assigned in a 2 × 2 factorial arrangement consisting of diet (control or 0.2% XPC) and inoculation (sterile broth or Salmonella). Pigs were fed a 3-phase nursery diet (0 to 7 d, 7 to 21 d, and 21 to 35 d) with ad libitum access to water and feed. On d 14, pigs were orally inoculated with 10(9) cfu of Salmonella enterica serovar Typhimurium DT104 or sterile broth. During d 17 to 20, all pigs were treated with a 5 mg/kg of BW intramuscular injection of ceftiofur-HCl. Growth performance and alterations in the gastrointestinal microbial ecology were measured during preinoculation (PRE; 0 to 14 d), sick (SCK; 14 to 21 d), and postinoculation (POST; 21 to 35 d). Body weight and ADG were measured weekly. Rectal temperature (RT) was measured weekly during PRE and POST, and every 12 h during SCK. Diet had no effect on BW, ADG, or RT during any period (P = 0.12 to 0.95). Inclusion of XPC tended (P < 0.10) to increase Salmonella shedding in feces during SCK. Consumption of XPC altered the composition of the gastrointestinal microbial community, resulting in increased (P < 0.05) populations of Bacteroidetes and Lactobacillus after Salmonella infection. Pigs inoculated with Salmonella had decreased ADG and BW, and increased RT during SCK (P < 0.001). Furthermore, fecal Salmonella cfu (log(10)) was modestly correlated (P = 0.002) with BW (r = -0.22), ADFI (r = -0.27), ADG (r = -0.36), G:F (r = -0.18), and RT (r = 0.52) during SCK. After antibiotic administration, all Salmonella-infected pigs stopped shedding. During POST, an interaction between diet and inoculation (P = 0.009) on ADG indicated that pigs infected with Salmonella grew better when eating XPC than the control diet. The addition of XPC to the diets of weanling pigs resulted in greater compensatory BW gains after infection with Salmonella than in pigs fed conventional nursery diets. This increase in BW gain is likely associated with an increase in beneficial bacteria within the gastrointestinal tract.

Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma.

BACKGROUND AND OBJECTIVES: Medullary thyroid carcinoma (MTC) occurs both sporadically and in the autosomal dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes. The distinction between true sporadic MTC and a new mutation familial case is important for future clinical management of both the patient and family. The susceptibility gene for MEN 2 is the RET proto-oncogene. Systematic analysis for germline mutations of the RET proto-oncogene was performed in a series of 67 patients with apparently sporadic MTC to determine whether they were true sporadic cases or unsuspected de novo MEN 2 cases. DESIGN AND PATIENTS: Sixty-seven unselected patients with sporadic MTC were randomly ascertained from clinic patients from four centres. The diagnosis of MTC was confirmed by histopathology. Germline DNA was extracted from peripheral blood leucocytes or from paraffin-embedded tissue and subsequently used for polymerase chain reaction amplification. MEASUREMENTS: Polymerase chain reaction based RET mutation analysis was performed by direct double-stranded cycle sequencing of exons 10, 11, 13 and 16, within which the majority of MEN2 mutations have been shown to occur. RESULTS: In this series, there was one proven case of germline mutation in RET codon 620, which previously has been shown to be responsible for MEN 2, thus indicating heritable disease. No germline mutation in codon 918, typical of MEN 2B, was found. CONCLUSIONS: A figure of 1.5% germline mutations in 67 apparently sporadic MTC is lower than the incidence of familial disease reported in previous series involving clinical and biochemical screening. The presence of a germline mutation in the RET proto-oncogene in a patient with MTC indicates heritable disease. The absence of germline RET exon 10, 11, 13 or 16 mutation appears to rule out MEN 2A to a high probability, although the presence of a familial form of MTC other than classical MEN 2A cannot be excluded conclusively.

A novel point mutation in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in a family with FMTC.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.

Familial site-specific ovarian cancer is linked to BRCA1 on 17q12-21.

In a study of nine families with "site-specific" ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age < 50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32-1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46-1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.

Haplotype analysis of MEN 2 mutations.

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome which affects thyroid C cells, and with variable frequency, the adrenal medulla, parathyroid and enteric autonomic ganglia. The syndrome is due to germline mutation in the receptor tyrosine kinase gene, RET. We have recently shown an unexpected correlation between one particular RET mutation, cys634-->arg, and the probability of parathyroid involvement in families with MEN 2A. Here we use haplotype analysis in the families to show that this correlation is not explained by a single founder chromosome which carries both the cys634-->arg mutation and a separate allele conferring susceptibility to parathyroid abnormality, but is probably due to the cys634-->arg mutation itself. The results also indicate that new mutations to MEN 2 are not infrequent.

Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours.

The susceptibility loci for the three multiple endocrine neoplasia (MEN) type 2 syndromes have been mapped to the region of chromosome 10q11.2 containing the RET proto-oncogene, which codes for a receptor tyrosine kinase. The majority of MEN 2A and familial medullary thyroid carcinoma results from missense mutations within one of five cysteine codons in the extracellular domain of the RET proto-oncogene. We now report a missense mutation, resulting in the substitution of a threonine for a methionine at codon 918 in the tyrosine kinase catalytic domain, in the germline of 26 of 28 apparently distinct families with MEN 2B. DNA from five of 13 apparently sporadic MTC and one of 12 apparently sporadic phaeochromocytomas harboured a similar mutation, but the corresponding germline DNA was wildtype in each case.

Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC.

We have analysed 118 families with inherited medullary thyroid carcinoma (MTC) for mutations of the RET proto-oncogene. These included cases of multiple endocrine neoplasia types 2A (MEN 2A) and 2B (MEN 2B) and familial MTC (FMTC). Mutations at one of 5 cysteines in the extracellular domain were found in 97% of patients with MEN 2A and 86% with FMTC but not in MEN 2B patients or normal controls. 84% of the MEN2A mutations affected codon 634. MEN 2A patients with a Cys634 to Arg substitution had a greater risk of developing parathyroid disease than those with other codon 634 mutations. Our data show a strong correlation between disease phenotype and the nature and position of the RET mutation, suggesting that a simple, constitutive activation of the RET tyrosine kinase is unlikely to explain the events leading to MEN 2A and FMTC.

An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes.

Neurofibromatosis (NF) type 1 (NF1) is notable for its variable expression. To determine whether variation in expression has an inherited component, we examined 175 individuals in 48 NF families, including six MZ twin pairs. Three quantitative traits were scored--number of café-au-lait patches, number of cutaneous neurofibromas, and head circumference; and five binary traits were scored--the presence or absence of plexiform neurofibromas, optic gliomas, scoliosis, epilepsy, and referral for remedial education. For café-au-lait patches and neurofibromas, correlation was highest between MZ twins, less high between first-degree relatives, and lower still between more distant relatives. The high correlation between MZ twins suggests a strong genetic component in variation of expression, but the low correlation between distant relatives suggests that the type of mutation at the NF1 locus itself plays only a minor role. All of the five binary traits, with the exception of plexiform neurofibromas, also showed significant familial clustering. The familial effects for these traits were consistent with polygenic effects, but there were insufficient data to rule out other models, including a significant effect of different NF1 mutations. There was no evidence of any association between the different traits in affected individuals. We conclude that the phenotypic expression of NF1 is to a large extent determined by the genotype at other "modifying" loci and that these modifying genes are trait specific.

Loss of NF1 alleles in phaeochromocytomas from patients with type I neurofibromatosis.

Type I neurofibromatosis (NF1) is a common autosomal dominant disorder that affects tissues derived from the neural crest. The manifestations are varied, comprising generalised disorders of growth and development as well as an increased risk of benign and malignant tumours including phaeochromocytomas and neurofibrosarcomas. The NF1 locus has been mapped to chromosome bands 17q11-12, and recently the NF1 gene has been cloned. Deletions identified in the constitutional genotype of some patients have suggested that the NF1 phenotype may arise from loss of function mutations of the NF1 gene, consistent with the hypothesis that it is a tumour suppressor gene. To date, however, analysis of NF1 tumours has not revealed the frequent allele losses encompassing the NF1 locus, implying loss of the wild-type NF1 allele, which would support this hypothesis. We report allele losses with markers flanking the NF1 region in each of 7 NF1 phaeochromocytomas. In each of the 3 tumours for which this could be determined, the loss involved the wild-type chromosome. These results provide strong evidence that, in cells of the adrenal medulla at least, the NFI gene may act as a tumour suppressor.

No evidence for germline mutations in exons 5-9 of the p53 gene in 25 breast cancer families.

Recent studies have demonstrated that families with the Li-Fraumeni syndrome carry inherited point mutations of the p53 gene. In the present study 25 families with strong histories of breast cancer were screened for the presence of such mutations. Polymerase chain reaction products of exons 5-9 of the p53 gene were examined by single-stranded conformational polymorphism analysis and, in addition, exon 7 was further screened by direct sequencing. No mutations were detected in constitutive DNA by either method. These results indicate that familial breast cancer does not usually result from germline point mutations in the p53 gene.

Mapping of von Hippel-Lindau disease to chromosome 3p confirmed by genetic linkage analysis.

Genetic linkage studies were performed in 12 British families with von Hippel-Lindau disease (VHL) using RFLPs at three loci (DNF15S2, THRB, RAF1) on the short arm of chromosome 3. Linkage was detected between the VHL disease locus and RAF1 with a maximum lod score of 3.88 at a recombination fraction of 0.05 (confidence interval 0.003-0.18). Multipoint linkage analysis suggested that the most likely location for the VHL disease locus is telomeric to THRB. These results confirm earlier reports localizing the VHL gene to the short arm of chromosome 3, and provide no evidence for genetic heterogeneity.

Paternal origin of new mutations in von Recklinghausen neurofibromatosis.

Von Recklinghausen neurofibromatosis (NF-1) is a common autosomal dominant disorder. The estimated new mutation rate (1 x 10(-4] is one of the highest for a human disorder. Here we report that in 12 of 14 families we have analysed, the new mutation is of paternal origin. This result is similar to that recently obtained for retinoblastoma. In other genetic disorders that show a bias towards paternal origin of new mutations, there is a marked increase in the incidence of mutations with paternal age, consistent with the mutations arising from replication errors in mitosis of spermatogonial stem cells. In retinoblastoma and NF-1, however, such paternal age effects are slight or absent. The mechanism or timing of germline mutation could therefore be different in the two cases.

The clinical and screening age-at-onset distribution for the MEN-2 syndrome.

The decision to screen for multiple endocrine neoplasia type 2 (MEN-2) is generally based on family history, the rationale for this approach being the presumed 100% penetrance of the disease. To determine the validity of this presumption we have estimated--by applying modifications of the life-table method--the clinical and screening age-at-diagnosis distributions for MEN-2, using families from the Cancer Research Campaign Medullary Thyroid Cancer Register and one large American family. The clinical penetrance of MEN-2 is shown to be incomplete, an estimated 41% of gene carriers not presenting with symptoms by age 70 on the basis of clinical history. Screening by the standard tests for detecting the earliest manifestations of the syndrome increases the penetrance to an estimated 93% by age 31. There is no evidence of a difference in the age-at-diagnosis distributions between maternal and paternal transmission, or among different families, but there is some suggestion of an earlier onset of medullary thyroid cancer in female gene carriers, and of a tendency of pheochromocytoma to cluster in families. These results can be used to calculate risks to relatives of affected individuals, which in turn can be used to guide decisions on which individuals to screen.