Recency-Weighted Statistical Modeling Approach to Attribute Illnesses Caused by 4 Pathogens to Food Sources Using Outbreak Data, United States.

Foodborne illness source attribution is foundational to a risk-based food safety system. We describe a method for attributing US foodborne illnesses caused by nontyphoidal Salmonella enterica, Escherichia coli O157, Listeria monocytogenes, and Campylobacter to 17 food categories using statistical modeling of outbreak data. This method adjusts for epidemiologic factors associated with outbreak size, down-weights older outbreaks, and estimates credibility intervals. On the basis of 952 reported outbreaks and 32,802 illnesses during 1998-2012, we attribute 77% of foodborne Salmonella illnesses to 7 food categories (seeded vegetables, eggs, chicken, other produce, pork, beef, and fruits), 82% of E. coli O157 illnesses to beef and vegetable row crops, 81% of L. monocytogenes illnesses to fruits and dairy, and 74% of Campylobacter illnesses to dairy and chicken. However, because Campylobacter outbreaks probably overrepresent dairy as a source of nonoutbreak campylobacteriosis, we caution against using these Campylobacter attribution estimates without further adjustment.

Salmonella Serotypes: A Novel Measure of Association with Foodborne Transmission.

Most nontyphoidal Salmonella (NTS) illnesses in the United States are thought to be foodborne. However, transmission routes likely vary among the different serotypes. We developed a relative ranking of NTS serotypes according to the strength of their association with foodborne transmission. We used Laboratory-based Enteric Disease Surveillance data to estimate the proportion of infections for each Salmonella serotype reported from 1998 to 2015 and Foodborne Disease Outbreak Surveillance System data to calculate the proportion of foodborne outbreak-associated Salmonella illnesses caused by each serotype. We calculated the ratios of these proportions to create a foodborne relatedness (FBR) measure for each serotype. Of the top 20 serotypes, Saintpaul (2.14), Heidelberg (1.61), and Berta (1.48) had the highest FBR measures; Mississippi (0.01), Bareilly (0.13), and Paratyphi B variant L(+) tartrate(+) (0.20) had the lowest. The FBRs for the three most prevalent serotypes were 1.22 for Enteritidis, 0.77 for Typhimurium, and 1.16 for Newport. This method provides a quantitative approach to estimating the relative differences in the likelihood that an illness caused by a particular serotype was transmitted by food, which may aid in tailoring strategies to prevent Salmonella illnesses and guide future research into serotype-specific source attribution.

Evaluation of the Use of Zero-Augmented Regression Techniques to Model Incidence of Campylobacter Infections in FoodNet.

The Foodborne Diseases Active Surveillance Network (FoodNet) is currently using a negative binomial (NB) regression model to estimate temporal changes in the incidence of Campylobacter infection. FoodNet active surveillance in 483 counties collected data on 40,212 Campylobacter cases between years 2004 and 2011. We explored models that disaggregated these data to allow us to account for demographic, geographic, and seasonal factors when examining changes in incidence of Campylobacter infection. We hypothesized that modeling structural zeros and including demographic variables would increase the fit of FoodNet's Campylobacter incidence regression models. Five different models were compared: NB without demographic covariates, NB with demographic covariates, hurdle NB with covariates in the count component only, hurdle NB with covariates in both zero and count components, and zero-inflated NB with covariates in the count component only. Of the models evaluated, the nonzero-augmented NB model with demographic variables provided the best fit. Results suggest that even though zero inflation was not present at this level, individualizing the level of aggregation and using different model structures and predictors per site might be required to correctly distinguish between structural and observational zeros and account for risk factors that vary geographically.

An Updated Scheme for Categorizing Foods Implicated in Foodborne Disease Outbreaks: A Tri-Agency Collaboration.

BACKGROUND: Foodborne disease data collected during outbreak investigations are used to estimate the percentage of foodborne illnesses attributable to specific food categories. Current food categories do not reflect whether or how the food has been processed and exclude many multiple-ingredient foods. MATERIALS AND METHODS: Representatives from three federal agencies worked collaboratively in the Interagency Food Safety Analytics Collaboration (IFSAC) to develop a hierarchical scheme for categorizing foods implicated in outbreaks, which accounts for the type of processing and provides more specific food categories for regulatory purposes. IFSAC also developed standard assumptions for assigning foods to specific food categories, including some multiple-ingredient foods. The number and percentage of outbreaks assignable to each level of the hierarchy were summarized. RESULTS: The IFSAC scheme is a five-level hierarchy for categorizing implicated foods with increasingly specific subcategories at each level, resulting in a total of 234 food categories. Subcategories allow distinguishing features of implicated foods to be reported, such as pasteurized versus unpasteurized fluid milk, shell eggs versus liquid egg products, ready-to-eat versus raw meats, and five different varieties of fruit categories. Twenty-four aggregate food categories contained a sufficient number of outbreaks for source attribution analyses. Among 9791 outbreaks reported from 1998 to 2014 with an identified food vehicle, 4607 (47%) were assignable to food categories using this scheme. Among these, 4218 (92%) were assigned to one of the 24 aggregate food categories, and 840 (18%) were assigned to the most specific category possible. CONCLUSIONS: Updates to the food categorization scheme and new methods for assigning implicated foods to specific food categories can help increase the number of outbreaks attributed to a single food category. The increased specificity of food categories in this scheme may help improve source attribution analyses, eventually leading to improved foodborne illness source attribution estimates and enhanced food safety and regulatory efforts.

Comparing Characteristics of Sporadic and Outbreak-Associated Foodborne Illnesses, United States, 2004-2011.

Outbreak data have been used to estimate the proportion of illnesses attributable to different foods. Applying outbreak-based attribution estimates to nonoutbreak foodborne illnesses requires an assumption of similar exposure pathways for outbreak and sporadic illnesses. This assumption cannot be tested, but other comparisons can assess its veracity. Our study compares demographic, clinical, temporal, and geographic characteristics of outbreak and sporadic illnesses from Campylobacter, Escherichia coli O157, Listeria, and Salmonella bacteria ascertained by the Foodborne Diseases Active Surveillance Network (FoodNet). Differences among FoodNet sites in outbreak and sporadic illnesses might reflect differences in surveillance practices. For Campylobacter, Listeria, and Escherichia coli O157, outbreak and sporadic illnesses are similar for severity, sex, and age. For Salmonella, outbreak and sporadic illnesses are similar for severity and sex. Nevertheless, the percentage of outbreak illnesses in the youngest age category was lower. Therefore, we do not reject the assumption that outbreak and sporadic illnesses are similar.

Quinolone-resistant Salmonella enterica serotype Enteritidis infections associated with international travel.

We found a strong association between nalidixic acid-resistant Salmonella enterica serotype Enteritidis infections in the United States and recent international travel by linking Salmonella Enteritidis data from the National Antimicrobial Resistance Monitoring System and the Foodborne Diseases Active Surveillance Network.

Source attribution of human salmonellosis: an overview of methods and estimates.

Reducing the burden of foodborne salmonellosis is challenging. It requires identification of the most important food sources causing disease and prioritization of effective intervention strategies. For this purpose, a variety of methods to estimate the relative contribution of different sources of Salmonella infections have been applied worldwide. Each has strengths and limitations, and the usefulness of each depends on the public health questions being addressed. In this study, we reviewed the source attribution methods and outcomes of several studies developed in different countries and settings, comparing approaches and regional differences in attribution estimates. Reviewed results suggest that illnesses and outbreaks are most commonly attributed to exposure to contaminated food, and that eggs, broiler chickens, and pigs are among the top sources. Although most source attribution studies do not attribute salmonellosis to produce, outbreak data in several countries suggest that exposure to raw vegetables is also an important source. International travel was also a consistently important exposure in several studies. Still, the relative contribution of specific sources to human salmonellosis varied substantially between studies. Although differences in data inputs, methods, and the point in the food system where attribution was estimated contribute to variability between studies, observed differences also suggest regional differences in the epidemiology of salmonellosis.

Outbreak-associated Salmonella enterica serotypes and food Commodities, United States, 1998-2008.

Salmonella enterica infections are transmitted not only by animal-derived foods but also by vegetables, fruits, and other plant products. To clarify links between Salmonella serotypes and specific foods, we examined the diversity and predominance of food commodities implicated in outbreaks of salmonellosis during 1998-2008. More than 80% of outbreaks caused by serotypes Enteritidis, Heidelberg, and Hadar were attributed to eggs or poultry, whereas >50% of outbreaks caused by serotypes Javiana, Litchfield, Mbandaka, Muenchen, Poona, and Senftenberg were attributed to plant commodities. Serotypes Typhimurium and Newport were associated with a wide variety of food commodities. Knowledge about these associations can help guide outbreak investigations and control measures.

Application of Bayesian techniques to model the burden of human salmonellosis attributable to U.S. food commodities at the point of processing: adaptation of a Danish model.

Mathematical models that estimate the proportion of foodborne illnesses attributable to food commodities at specific points in the food chain may be useful to risk managers and policy makers to formulate public health goals, prioritize interventions, and document the effectiveness of mitigations aimed at reducing illness. Using human surveillance data on laboratory-confirmed Salmonella infections from the Centers for Disease Control and Prevention and Salmonella testing data from U.S. Department of Agriculture Food Safety and Inspection Service's regulatory programs, we developed a point-of-processing foodborne illness attribution model by adapting the Hald Salmonella Bayesian source attribution model. Key model outputs include estimates of the relative proportions of domestically acquired sporadic human Salmonella infections resulting from contamination of raw meat, poultry, and egg products processed in the United States from 1998 through 2003. The current model estimates the relative contribution of chicken (48%), ground beef (28%), turkey (17%), egg products (6%), intact beef (1%), and pork (<1%) across 109 Salmonella serotypes found in food commodities at point of processing. While interpretation of the attribution estimates is constrained by data inputs, the adapted model shows promise and may serve as a basis for a common approach to attribution of human salmonellosis and food safety decision-making in more than one country.

Duck hunters' perceptions of risk for avian influenza, Georgia, USA.

To determine duck hunters'risk for highly pathogenic avian influenza, we surveyed duck hunters in Georgia, USA, during 2007-2008, about their knowledge, attitudes, and practices. We found they engage in several practices that could expose them to the virus. Exposures and awareness were highest for those who had hunted >10 years.

Effect of Salmonella vaccination of breeder chickens on contamination of broiler chicken carcasses in integrated poultry operations.

While measures to control carcass contamination with Salmonella at the processing plant have been implemented with some success, on-farm interventions that reduce Salmonella prevalence in meat birds entering the processing plant have not translated well on a commercial scale. We determined the impact of Salmonella vaccination on commercial poultry operations by monitoring four vaccinated and four nonvaccinated breeder (parental) chicken flocks and comparing Salmonella prevalences in these flocks and their broiler, meat bird progeny. For one poultry company, their young breeders were vaccinated by using a live-attenuated Salmonella enterica serovar Typhimurium vaccine (Megan VAC-1) followed by a killed Salmonella bacterin consisting of S. enterica serovar Berta and S. enterica serovar Kentucky. The other participating poultry company did not vaccinate their breeders or broilers. The analysis revealed that vaccinated hens had a lower prevalence of Salmonella in the ceca (38.3% versus 64.2%; P < 0.001) and the reproductive tracts (14.22% versus 51.7%; P < 0.001). We also observed a lower Salmonella prevalence in broiler chicks (18.1% versus 33.5%; P < 0.001), acquired from vaccinated breeders, when placed at the broiler farms contracted with the poultry company. Broiler chicken farms populated with chicks from vaccinated breeders also tended to have fewer environmental samples containing Salmonella (14.4% versus 30.1%; P < 0.001). There was a lower Salmonella prevalence in broilers entering the processing plants (23.4% versus 33.5%; P < 0.001) for the poultry company that utilized this Salmonella vaccination program for its breeders. Investigation of other company-associated factors did not indicate that the difference between companies could be attributed to measures other than the vaccination program.

Distribution, diversity, and seasonality of waterborne salmonellae in a rural watershed.

Salmonella outbreaks from contaminated water and nonanimal foods (e.g., produce) are increasingly reported. To address the environment as a potential source of pathogenic Salmonella, we investigated levels of salmonellae and the geographic and temporal variation of Salmonella serotypes from surface waters in a region of Georgia (United States) with a history of high salmonellosis case rates. Monthly water samples were collected from six stations in the Little River (Upper Suwannee Basin) for 12 months (April 2005 to April 2006). Salmonellae were enumerated using a three-step most-probable-number (MPN) assay. Salmonellae were detected in 57 of the 72 water samples collected (79.2%). Monthly Salmonella densities ranged from an MPN of 2.5 liter(-1) in April 2005 to 36.3 liter(-1) in August 2005; concentrations were significantly higher in the summer months compared to other seasons (P<0.05). Concentrations were not significantly different between stations. Levels of salmonellae were correlated with average daily watershed rainfall for the 1 and 2 days preceding each sample collection (r=0.77 and 0.68, respectively; P<0.005). Additionally, water temperature was also positively associated with total Salmonella levels (r=0.44; P<0.05). In total, 13 S. enterica serotypes were identified among 197 Salmonella isolates. Eighty (40.6%) were identified as S. enterica subsp. arizonae. Muenchen and Rubislaw were the most frequently identified serotypes of the remaining 117 isolates (28 and 26 isolates, respectively). Serotype diversity peaked in the summer, with 9 serotypes observed in August compared to only one serotype (S. enterica subsp. arizonae) observed in April (2005 and 2006) (P<0.05). Furthermore, all samples collected in August (6/6) contained multiple serotypes (two to five per sample). The results of this study suggest that Salmonella abundance and diversity in the environment vary temporally and are strongly influenced by seasonal precipitation and water temperature.

Human contacts and potential pathways of disease introduction on Georgia poultry farms.

As highly pathogenic avian influenza H5N1 virus continues to circulate in the world, poultry farm biosecurity and timely reporting of morbidity and mortality among commercial poultry farms in the United States are major concerns. To assess the vulnerability of poultry farms to the introduction and spread of a highly infectious pathogen, such as the currently circulating H5N1 influenza virus, a survey was administered to growers in two counties in Georgia representing areas of low and high poultry densities. Survey questions regarding horizontal contacts and management were sent to commercial broiler and breeder-layer chicken producers. Responses were used to estimate and compare contact rates and patterns between the two regions. The distribution of high-risk visitors (i.e., those going inside the poultry houses) to poultry farms did not vary significantly between growers in counties with high and low poultry densities or between breeder-layer and broiler growers. Compared with broiler producers in the county with high poultry density, broiler growers in the county with low poultry density were more likely to hire non-family employees to help with poultry management (62% vs. 17%; P = 0.001) and assist other growers with their poultry (31% vs. 6%; P = 0.025). Use of contracted litter services was significantly higher (P = 0.019) among broiler growers in the poultry-dense county (40%) compared with the low-density county (6%). Compared with broiler growers, breeder-layer producers also were significantly more likely to hire non-family employees to help on the farm (53% vs. 17%; P = 0.008). Poultry growers in the highly poultry-dense county were more likely to have a public road or field receiving poultry litter within a quarter mile of their poultry houses, compared with those in the lower density county. Data obtained in this study support the observations of published poultry disease outbreak investigations and highlight the differences in farm vulnerability to disease introduction within areas of different poultry densities and management practices.

Genetic Mutations in B-Acute Lymphoblastic Leukemia Among African American and European American Children.

BACKGROUND: The survival of patients with B-acute lymphoblastic leukemia (B-ALL) is significantly lower in African American (AA) children compared with European American children (EA). Here, we present a whole exome sequencing (WES) study showing race-specific genetic variations that may play a role on the disparate outcomes among AA and EA children with B-ALL. PATIENTS AND METHODS: Five AA and 15 EA patients ranging in age from 1 to 18 years were enrolled. The median blast percentage was 94.8% (range, 64.5%-99.9%). Frozen bone marrow aspirate was used to extract DNA, and WES was performed, focusing on race and B-ALL-specific germline mutations. RESULTS: Most genetic variants (n = 339) were shared between AA and EA children. Some genetic aberrations were only uniquely identified in AA (n = 58) and others in EA (n = 52) In AA, the genetic aberrations clustered in canonical pathways related to telomerase signaling and cancer signaling. In EA, the unique genetic aberration clustered in pathways related to stem cell pluripotency and hereditary cancer. CONCLUSIONS: Our study revealed aberrant genetic aberrations in signaling networks that may contribute to race-specific aspects of leukemogenesis. Our results suggest the value of WES as a tool for development of individual gene signatures and gene scores for AA and EA children afflicted by B-ALL. These findings may ultimately impact disease management and contribute to the elimination of disparate outcomes in AA children with B-ALL.

Prevalence of Mycoplasma bovis in backgrounding and stocker cattle operations.

OBJECTIVE: To characterize the prevalence of Mycoplasma bovis infection in backgrounding and stocker cattle operations and compare bacteriologic culture with PCR assay for detection of M bovis. DESIGN: Prospective descriptive study. ANIMALS: 432 calves, 3 to 9 months old, from 9 operations. PROCEDURES: 2 nasal swab specimens were collected from each calf. Swab specimens were evaluated via bacteriologic culture and PCR assay for organisms of the class Mollicutes and M bovis. Culture results were considered negative if no growth occurred within 21 days. Positive results were indicated by characteristic colony formation with PCR assay confirmation. Deoxyribonucleic acid was extracted from 1 swab specimen for direct PCR assay for Mollicutes and M bovis. RESULTS: Of 432 calves, 374 (87%) had positive results for Mollicutes via PCR assay and 63 (15%) via culture. Seven (2%) calves had positive results for M bovis via PCR assay and 10 (2%) via culture. Prevalence of Mollicutes at the farm level ranged from 54% to 100% via PCR assay and from 0% to 59% via culture. Prevalence of M bovis at the farm level ranged from 0% to 4% via PCR assay and from 0% to 6% via culture. Calves that shed M bovis were significantly more likely to have a fever than were calves that did not shed M bovis. CONCLUSIONS AND CLINICAL RELEVANCE: M bovis was detected at a low level in recently purchased backgrounded and stocker calves in Georgia. Although slightly more infected calves were detected via culture and PCR assay together, PCR assay appeared to accurately identify M bovis at the farm level.

Bioterrorism with zoonotic disease: public health preparedness lessons from a multiagency exercise.

Responding to agricultural bioterrorism with pathogenic agents that are communicable from animals to humans (zoonotic diseases) requires effective coordination of many organizations, both inside and outside of government. Action must be simultaneously taken to address public health concerns, respond to the agricultural dimensions of the event, and carry out the necessary law enforcement investigation. As part of a project focused on examining public health preparedness in Georgia, an exercise was carried out in July 2005 examining the intentional introduction of avian influenza (H5N1) in commercial poultry operations. The attack scenario, which was written to occur during an already severe human influenza season, enabled exploration of a range of issues associated with public health preparedness for major disease outbreaks including pandemic influenza, coordination of a multiagency response operation at multiple levels of government, and effective management of interdisciplinary response activities. The exercise is described and broader policy lessons regarding preparedness planning are discussed.

Risk factors associated with renal insufficiency in horses with primary gastrointestinal disease: 26 cases (2000-2003).

OBJECTIVE: To identify factors associated with renal insufficiency in colic- or colitis-affected horses with high serum creatinine (SCr) concentrations evaluated at a referral hospital. DESIGN: Retrospective case series. ANIMALS: 167 colic- or colitis-affected horses (88 represented a random sample [hospital population], and 79 had high SCr concentration at initial evaluation [study population]). PROCEDURE: Medical records were reviewed. Data collected included signalment; physical examination, clinicopathologic, and diagnostic findings; and outcome. The study population was categorized on the basis of whether SCr concentration did (AR group; n = 53) or did not (PA group; 26) normalize within 72 hours of fluid therapy. Characteristics of the study and hospital populations were compared. RESULTS: Males and Quarter Horses were significantly overrepresented in the study population. Compared with the hospital population, study-population horses were significantly more likely to have colitis, gastric reflux, and diarrhea at initial evaluation. Initial mean SCr concentration in the PA group was significantly higher than the AR group; identification of gastric reflux, abnormal rectal examination findings, and hypochloremia were significantly associated with persistent azotemia after 72 hours of fluid therapy. Compared with the AR group, PA group horses were 3 times as likely to die or be euthanized. CONCLUSIONS AND CLINICAL RELEVANCE: In colic- or colitis-affected horses, factors associated with renal insufficiency included gastric reflux, abnormal rectal examination findings, or hypochloremia initially; prognosis for horses in which azotemia resolves within 72 hours of treatment appears to be better than for horses with persistent azotemia.

Diversity and Persistence of Salmonella enterica Strains in Rural Landscapes in the Southeastern United States.

Salmonellosis cases in the in the United States show distinct geographical trends, with the southeast reporting among the highest rates of illness. In the state of Georgia, USA, non-outbreak associated salmonellosis is especially high in the southern low-lying coastal plain. Here we examined the distribution of Salmonella enterica in environmental waters and associated wildlife in two distinct watersheds, one in the Atlantic Coastal Plain (a high case rate rural area) physiographic province and one in the Piedmont (a lower case rate rural area). Salmonella were isolated from the two regions and compared for serovar and strain diversity, as well as distribution, between the two study areas, using both a retrospective and prospective design. Thirty-seven unique serovars and 204 unique strain types were identified by pulsed-field gel electrophoresis (PFGE). Salmonella serovars Braenderup, Give, Hartford, and Muenchen were dominant in both watersheds. Two serovars, specifically S. Muenchen and S. Rubislaw, were consistently isolated from both systems, including water and small mammals. Conversely, 24 serovars tended to be site-specific (64.8%, n = 37). Compared to the other Salmonella serovars isolated from these sites, S. Muenchen and S. Rubislaw exhibited significant genetic diversity. Among a subset of PFGE patterns, approximately half of the environmental strain types matched entries in the USA PulseNet database of human cases. Ninety percent of S. Muenchen strains from the Little River basin (the high case rate area) matched PFGE entries in PulseNet compared to 33.33% of S. Muenchen strains from the North Oconee River region (the lower case rate area). Underlying the diversity and turnover of Salmonella strains observed for these two watersheds is the persistence of specific Salmonella serovars and strain types that may be adapted to these watersheds and landscapes.

Methodological Framework for World Health Organization Estimates of the Global Burden of Foodborne Disease.

BACKGROUND: The Foodborne Disease Burden Epidemiology Reference Group (FERG) was established in 2007 by the World Health Organization to estimate the global burden of foodborne diseases (FBDs). This paper describes the methodological framework developed by FERG's Computational Task Force to transform epidemiological information into FBD burden estimates. METHODS AND FINDINGS: The global and regional burden of 31 FBDs was quantified, along with limited estimates for 5 other FBDs, using Disability-Adjusted Life Years in a hazard- and incidence-based approach. To accomplish this task, the following workflow was defined: outline of disease models and collection of epidemiological data; design and completion of a database template; development of an imputation model; identification of disability weights; probabilistic burden assessment; and estimating the proportion of the disease burden by each hazard that is attributable to exposure by food (i.e., source attribution). All computations were performed in R and the different functions were compiled in the R package 'FERG'. Traceability and transparency were ensured by sharing results and methods in an interactive way with all FERG members throughout the process. CONCLUSIONS: We developed a comprehensive framework for estimating the global burden of FBDs, in which methodological simplicity and transparency were key elements. All the tools developed have been made available and can be translated into a user-friendly national toolkit for studying and monitoring food safety at the local level.

Comparison of different tests to diagnose feline infectious peritonitis.

Clinical data from 488 cats (1979-2000) with histopathologically confirmed feline infectious peritonitis (FIP) and 620 comparable controls were evaluated retrospectively to assess the value of several diagnostic tests frequently used in the evaluation of cats with suspected FIP. Diagnostic utility of serum albumin to globulin ratio for the diagnosis of FIP was greater than of the utility of serum total protein and gamma-globulin concentrations. Diagnostic utility of these variables was higher when performed on effusion. On effusion, positive and negative predictive values of Rivalta's test, a test that distinguishes between exudates and transudates (0.86 and 0.97), anti-coronavirus antibody detection (0.90 and 0.79), and immunofluorescence staining of coronavirus antigen in macrophages (1.00 and 0.57) were investigated. The positive and negative predictive values of presence of anti-coronavirus antibodies were 0.44 and 0.90, respectively, antibody concentrations (1:1,600) were 0.94 and 0.88. presence of immune complexes measured by a competitive enzyme-linked immunosorbent assay were 0.67 and 0.84, and detection of viral RNA by serum reverse-transcriptase polymerase chain reaction (RT-PCR) were 0.90 and 0.47. Effusion RT-PCR was performed in 6 cats; it was positive in all 5 cats with FIP and negative in the cat with another disease. Diagnostic assays on the fluid in cats with body effusion had good predictive values. Definitive diagnosis of FIP on the basis of measurement of various variables in serum was not possible. Serum tests can only be used to facilitate the decision for more invasive diagnostic methods.