The association between oil spill cleanup-related total hydrocarbon exposure and diabetes.

BACKGROUND: Although evidence suggests relationships between some crude oil components and glycemic dysregulation, no studies have examined oil spill-related chemical exposures in relation to type 2 diabetes mellitus (DM) risk. This study examined the relationship between total hydrocarbon (THC) exposure among workers involved in the 2010 Deepwater Horizon (DWH) oil spill and risk of DM up to 6 years afterward. METHODS: Participants comprised 2660 oil-spill cleanup or response workers in the prospective GuLF Study who completed a clinical exam and had no self-reported DM diagnosis prior to the spill. Maximum THC exposure was estimated with a job-exposure matrix based on interview data and personal measurements taken during cleanup operations. We defined incident DM by self-reported physician diagnosis of DM, antidiabetic medication use, or a measured hemoglobin A1c value ≥ 6.5%. We used log binomial regression to estimate risk ratios (RRs) for DM across ordinal categories of THC exposure. The fully adjusted model controlled for age, sex, race/ethnicity, education, employment status, and health insurance status. We also stratified on clinical body mass index categories. RESULTS: We observed an exposure-response relationship between maximum daily ordinal THC exposure level and incident DM, especially among overweight participants. RRs among overweight participants were 0.99 (95% CI: 0.37, 2.69), 1.46 (95% CI: 0.54, 3.92), and 2.11 (95% CI: 0.78, 5.74) for exposure categories 0.30-0.99 ppm, 1.00-2.99 ppm, and ≥3.00 ppm, respectively (ptrend = 0.03). CONCLUSION: We observed suggestively increasing DM risk with increasing THC exposure level among overweight participants, but not among normal weight or obese participants.

Dietary intake of flavonoids and oesophageal and gastric cancer: incidence and survival in the United States of America (USA).

BACKGROUND: Flavonoids, polyphenolic compounds concentrated in fruits and vegetables, have experimentally demonstrated chemopreventive effects against oesophageal and gastric cancer. Few epidemiologic studies have examined flavonoid intake and incidence of these cancers, and none have considered survival. METHODS: In this USA multicentre population-based study, case participants (diagnosed during 1993-1995 with oesophageal adenocarcinoma (OEA, n=274), gastric cardia adenocarcinoma (GCA, n=248), oesophageal squamous cell carcinoma (OES, n=191), and other gastric adenocarcinoma (OGA, n=341)) and frequency-matched controls (n=662) were interviewed. Food frequency questionnaire responses were linked with USDA Flavonoid Databases and available literature for six flavonoid classes and lignans. Case participants were followed until 2000 for vital status. Multivariable-adjusted odds ratios (ORs) and hazard ratios (HRs) (95% confidence intervals (CIs)) were estimated, comparing highest with lowest intake quartiles, using polytomous logistic and proportional hazards regressions, respectively. RESULTS: Little or no consistent association was found for total flavonoid intake (main population sources: black tea, orange/grapefruit juice, and wine) and incidence or survival for any tumour type. Intake of anthocyanidins, common in wine and fruit juice, was associated with a 57% reduction in the risk of incident OEA (OR=0.43, 95% CI=0.29-0.66) and OES (OR=0.43, 95% CI=0.26-0.70). The ORs for isoflavones, for which coffee was the main source, were increased for all tumours, except OES. Anthocyanidins were associated with decreased risk of mortality for GCA (HR=0.63, 95% CI=0.42-0.95) and modestly for OEA (HR=0.87, 95% CI=0.60-1.26), but CIs were wide. CONCLUSIONS: Our findings, if confirmed, suggest that increased dietary anthocyanidin intake may reduce incidence and improve survival for these cancers.

Neurologic symptoms in licensed pesticide applicators in the Agricultural Health Study.

Exposure to high levels of many pesticides has both acute and long-term neurologic consequences, but little is known about the neurotoxicity of chronic exposure to moderate pesticide levels. We analysed cross-sectional data from 18 782 Caucasian, male, licensed pesticide applicators, enrolled in the Agricultural Health Study from 1993 to 1997. Applicators provided information on lifetime pesticide use, and 23 neurologic symptoms typically associated with pesticide intoxication. Increased risk of experiencing >/=10 symptoms during the year before enrollment was associated with cumulative pesticide use, personally mixing or applying pesticides, pesticide-related medical care, diagnosed pesticide poisoning, and events involving high personal pesticide exposure. Greatest risk was associated with use of organophosphate and organochlorine insecticides. Results were similar after stratification by pesticide use during the year before enrollment, or exclusion of applicators with a history of pesticide poisoning, or high-exposure events. Use of pesticide application methods likely to involve high personal exposure was associated with greater risk. Groups of symptoms reflecting several neurologic domains, including affect, cognition, autonomic and motor function, and vision, were also associated with pesticide exposure. These results suggest that neurologic symptoms are associated with cumulative exposure to moderate levels of organophosphate and organochlorine insecticides, regardless of recent exposure or history of poisoning.

Validity study of self-reported pesticide exposure among orchardists.

Self-reported work histories are often the only means of estimating occupational exposures in epidemiologic research. The objective of this study was to examine the accuracy of recall of historical pesticide use among orchardists. All 185 orchardists in this study had participated previously in a cohort study of men occupationally exposed to pesticides. In that study (1972 to 1976), subjects were interviewed annually and asked to list pesticides used since the last interview. In 1997, 265 of the 440 presumed-living orchardists from the original cohort were successfully recontacted and asked to complete a detailed questionnaire concerning their lifetime use of pesticides; 185 (69.8% of farmers successfully contacted) agreed. Considering the 1972-1976 data as the standard, sensitivity and specificity of recall were calculated for certain pesticides and pesticide categories. Sensitivity of recall was good to excellent (0.6-0.9) for the broad categories of insecticides, herbicides, and fungicides, for heavily used chemical classes, such as organophosphates and organochlorines, and for commonly used pesticides; it was lower and more variable (0.1-0.6) for specific pesticides. Recall specificity was greatest (0.7-0.9) for the least used pesticides and chemical classes, such as dithiocarbamates and manganese-containing pesticides, and was generally modest for the rest (0.5-0.6). There was no evidence of selection bias between study participants and nonparticipants. In conclusion, recall accuracy was good for commonly used pesticides and pesticide categories. This level of recall accuracy is probably adequate for epidemiologic analyses of broad categories of pesticides, but is a limitation for detecting more specific associations.

Glutathione S-transferase genotypes and stomach cancer in a population-based case-control study in Warsaw, Poland.

Glutathione S-transferases are important in the detoxification of a wide range of human carcinogens. Previous studies have shown inconsistent associations between the GSTT1 and GSTM1 null genotypes and stomach cancer risk. We investigated the relationship between these and related genotypes and stomach cancer risk in a population-based case-control study in Warsaw, Poland, where stomach cancer incidence and mortality rates are among the highest in Europe. DNA from blood samples was available for 304 stomach cancer patients and 427 control subjects. We observed a 1.48-fold increased risk for stomach cancer (95% confidence interval 0.97-2.25) in patients with the GSTT1 null genotype but no evidence of increased risk associated with the GSTM1, GSTM3 or GSTP1 genotypes. Furthermore, the stomach cancer risk associated with the GSTT1 null genotype varied by age at diagnosis, with odds ratios of 3.85, 1.91, 1.78 and 0.59 for those diagnosed at ages less than 50, 50-59, 60-69 and 70 years or older, respectively (P trend = 0.01). This was due to a shift in the GSTT1 genotype distribution across age groups among stomach cancer patients only. These results suggest that the GSTT1 null genotype may be associated with increased risk of stomach cancer.

Development of a life events/icon calendar questionnaire to ascertain occupational histories and other characteristics of migrant farmworkers.

BACKGROUND: Specialized methods are necessary to collect data from migrant farmworkers for epidemiologic research. METHODS: We developed a questionnaire that collected lifetime occupational histories and other lifestyle risk factors via a life events/icon calendar, and administered the questionnaire to a convenience sample of 162 migrant farmworkers in nine areas of the U.S. RESULTS: The average duration of the interviews was about 1 h 30 min, with an average of 45 min for the work history section. The occupational histories covered a median of 27.6 years per person for men and 20.8 years per person for women. The median number of years spent in farm jobs was 11.3 for men and 5.8 for women. The median number of farm jobs (crop/task combination) per person was 59 among men and 27 among women. Many farmworkers performed the same crop/task combinations at multiple times throughout their lives, yielding a median of 13 unique farm jobs and 8 unique crops among men and 7 jobs and 5 crops among women. CONCLUSIONS: The project demonstrated that it is feasible to collect detailed work histories and other risk factor data from farmworkers, documented the complexity of work histories encountered among farmworkers, and yielded recommendations for refining a questionnaire that will facilitate future epidemiologic research on farmworkers.

Test-retest reliability of an icon/calendar-based questionnaire used to assess occupational history.

BACKGROUND: Self-reported work histories can be critical for both epidemiologic and clinical purposes. However, the complexity of some work histories, such as those of migrant farm workers, can hamper recall, resulting in inaccurate reporting. Memory aids may reduce such error. This study assesses the reliability of work histories collected using such aids in the form of an icon/calendar-based questionnaire. METHODS: Thirty-one males engaged in farmwork and other manual labor for a median 28 years (range: 10-64) were interviewed twice, 8-14 months apart, about their lifetime employment. In each interview, subjects were asked about important life events, which were recorded with icons on a calendar. They were then asked to recount their work history, including for each job the tasks, crops or products handled, starting and ending dates, and location. This information was recorded, job-by-job, on the calendar with icons. RESULTS: Interquestionnaire agreement of cumulative reported employment duration (as measured by the correlation coefficient) was moderate to high across all time periods for certain crops (e.g., r = 0.69-0.92 for apple-related work), by location (e.g., r = 0.76-0.95 for Washington State), and for agricultural work in general (r = 0.67-0.94), but was lower for specific tasks. Agreement of job counts was high for total work history for certain crops (e.g., r = 0.93 for apple-related work), by location (e.g., r = 0.90 for Washington State), and for agricultural work in general (r = 0.89), but paradoxically decreased with proximity to the interview date. Agreement of both measures tended to be highest for those tasks and crops in which subjects reported spending the most time. Categorization of subjects into tertiles on the basis of either cumulative duration or counts produced results similar to those observed for job counts. CONCLUSIONS: The icon-calendar questionnaire is an effective tool for estimating cumulative duration of certain work categorizations among subjects with complex work histories.

Comparison of a traditional questionnaire with an icon/calendar-based questionnaire to assess occupational history.

BACKGROUND: Self-reported work histories are an essential tool for estimating exposure in many occupational epidemiologic studies. However, the transience of some occupations such as farm work can hamper recall, resulting in inaccurate reporting. To address this problem, we have developed an icon/calendar-based questionnaire. This study compares work histories collected via this questionnaire to those collected via a traditional questionnaire. METHODS: Eighty-nine farmworkers and non-farmworkers were interviewed twice, 8-10 months apart, about their lifetime employment. In the first interview, subjects were asked to recount their entire work history, starting from the interview date and moving backwards in time ("traditional questionnaire"). In the second interview, subjects were first asked about important life events, which were recorded with icons on a calendar. They were then asked to recount their work history, which was recorded, job-by-job, on the calendar with icons ("icon-calendar questionnaire"). RESULTS: Number of jobs and amount of work time accounted for since first employment were significantly greater using the icon-calendar questionnaire than the traditional questionnaire, the disparity increasing with time from the date of interview. The ratio of number of jobs in the traditional questionnaire to number of jobs in the icon-calendar questionnaire decreased from 100.0% in the most recent time period to 0.0% in the earliest time period. While the percentage of time explained by employment remained relatively constant across time periods in the icon-calendar questionnaire, ranging from 86.3 to 98.9%, it rapidly decreased with time in the traditional questionnaire, from 77.9% in the most recent time period to 0.0% in the earliest time period. CONCLUSIONS: The icon-calendar questionnaire was more effective than the traditional questionnaire for obtaining complex work histories during interviews, producing a more complete picture of a person's work history.

Comparability of data obtained from migrant farmworkers and their spouses on occupational history.

BACKGROUND: Epidemiologic studies, particularly case-control studies, often rely on proxy respondents to provide information about subjects' occupational histories. The quality of proxy-reported information in occupational histories has never been evaluated for migrant farmworkers. METHODS: We compared occupational histories self-reported by 31 farmworkers with those reported by their wives. The work histories were obtained using an icon/calendar-based questionnaire that was designed to facilitate recall for migrant farmworkers, who typically have complex work histories. RESULTS: The work histories provided by proxy respondents contained 32% fewer jobs and accounted for 24% fewer years than the self-reported histories. Correlations for lifetime duration of employment in different types of jobs were moderate to good for general agricultural jobs in Washington (0.70) and apple-related jobs (0.65), which were held by virtually all of the farmworkers; correlations were moderate to poor for less common jobs and for specific types of tasks. Agreement was better after marriage than before, and for jobs held in the current year compared to other time frames. Overall, the ability of the spouses to provide occupational histories for farmworkers was within the range observed in studies involving other occupations and industries. CONCLUSIONS: In studies involving farmworkers, when study subjects cannot be interviewed, spouses can provide useful information on occupational histories. However, the information should be used only for more generalized exposure assessments; it is most appropriate for estimating cumulative duration of agricultural work, or recent work, by place or for common crops.

National farmworker database: establishing a farmworker cohort for epidemiologic research.

BACKGROUND: There is little research into the long-term health effects of pesticides and other agricultural exposures among seasonal and migrant farmworkers in the United States. We present results of a feasibility study that established a cohort of farmworkers for use in epidemiologic research. METHODS: Subjects consisted of migrant and seasonal farmworkers who joined the cohort while seeking social services through members of the Association of Farmworker Opportunity Programs (AFOP) and were entered in the National Farmworker Database (NFD) between the end of 1997 and March 1999. We designed an add-on interview with information that enhanced the utility of the database for epidemiologic research. RESULTS: We recruited and obtained basic demographic and employment information on 5,597 farmworkers at very modest cost and effort. Subjects were mostly seasonal (61.5%), female (56.7%), and Hispanic (67.4%), with a median age of 27. Most (62.6%) had not completed high school; almost all (99.1%) reported being U.S. citizens or permanent residents, an eligibility requirement for some of the services provided by AFOP. The majority (62.5%) had engaged in farmwork for less than 10 years, but had performed a wide variety of tasks on different crops, including row crops and tree fruits. Picking was the most common task reported. Most subjects had performed farmwork in Florida, North Carolina, Texas, Michigan, or Georgia. For usual source of health care, 63.7% reported use of U.S. hospitals or emergency rooms/clinics, 42.0% U.S. private physicians, and 29.7% migrant health clinics. Among subjects reporting a prior diagnosis of cancer, primary sources of health care for treatment of that cancer included U.S. private physicians (61.9%), U.S. hospitals or emergency rooms/clinics (23.8%), and migrant health clinics (10.5%). CONCLUSIONS: Results suggest that by adding a brief interview to the existing NFD data collection process, which was designed for other purposes, it is feasible to create an efficient tool for conducting longitudinal epidemiologic research among farmworkers.

Parkinsonism and occupational exposure to pesticides.

OBJECTIVE: To examine the risk of parkinsonism related to lifetime occupational exposure to pesticides among a cohort of men, mostly orchardists, in Washington State. METHODS: All 310 subjects in this study had previously participated in a cohort study of men occupationally exposed to pesticides. Subjects were given a structured neurological examination and completed a self administered questionnaire which elicited detailed information on pesticide (insecticide, herbicide, and fungicide) use throughout their working careers. Demographic characteristics were also sought. Subjects had a mean age of 69.6 years (range 49-96, SD 8.1). There were 238 (76.8%) subjects who reported some occupational exposure to pesticides, whereas 72 (23.2%) reported none. Parkinsonism was defined by the presence of two or more of rest tremor, rigidity, bradykinesia, and impairment of postural reflexes in subjects not on antiparkinsonian medication, or the presence of at least one sign if they were on such medication. Parkinson's disease was not studied explicitly because of the difficulty in distinguishing it from other parkinsonian syndromes. A generalised linear model was used to estimate prevalence ratios (PRs) for parkinsonism relative to history of farming, pesticide use, and use of well water. RESULTS: A PR of 2.0 (95% confidence interval (95% CI) 1.0 to 4.2) was found for subjects in the highest tertile of years of exposure to pesticides; a similarly increased, non-significant, PR was found for the middle tertile (1.9 (95% CI 0.9 to 4.0)), although a trend test did not show a significant exposure-response relation. No increased risks were found associated with specific pesticides or pesticide classes, nor with a history of farming or use of well water. CONCLUSION: Parkinsonism may be associated with long term occupational exposure to pesticides, although no associations with specific pesticides could be detected. This finding is consistent with most of the publications on this topic.

Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases.

Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.

Maternal occupation in agriculture and risk of limb defects in Washington State, 1980-1993.

OBJECTIVES: This study examined the association between maternal occupational exposure to agricultural chemicals and the risk of limb defects among offspring. METHODS: A retrospective cohort study was conducted using Washington State birth records for the years 1980 through 1993. The exposed group, consisting of 4466 births to mothers employed in agriculture, was compared with 2 reference groups: (i) 23,512 births in which neither parent worked in agriculture ("nonagricultural" group) and (ii) 5994 births in which only the father worked in agriculture ("paternal agriculture" group). The outcome of interest was limb defects [syndactyly, polydactyly, adactyly, and "other limb reductions" (as listed in the birth record)]. RESULTS: An elevated risk of limb defects was observed for the exposed group in comparison with both the nonagricultural and paternal agriculture groups, with ethnicity-adjusted prevalence ratios of 2.6 [95% confidence interval (95% CI) 1.1-5.8] and 2.6 (95% CI 0.7-9.5), respectively. CONCLUSIONS: These results support the hypothesis that maternal occupational exposure to agricultural chemicals may increase the risk of giving birth to a child with limb defects.

Serratia marcescens keratitis: strain-specific corneal pathogenesis in rabbits.

PURPOSE: The purpose of this study was to develop an animal model of Serratia keratitis that is suitable to demonstrate the pathology of specific strains. METHODS: Serratia marcescens ocular strains 93-1399-1 and 94-EI-185-2, and an environmental strain (ATCC 14041) were characterized in vitro in terms of their motility, metabolic profiles, ribotypes, and protease production. The strains were then analyzed in the rabbit intrastromal injection model. Slit lamp examination (SLE) and enumeration of bacteria in the cornea was conducted every 6 hours for 30 hours post-infection. In vivo motilities were analyzed by quantification of bacteria in the peripheral and central areas of infected rabbit corneas. RESULTS: All strains were similar in their metabolic activity and production of extracellular proteases. The ocular isolates were distinct from the environmental strain in their ribotyping patterns and in their motility. Each strain grew logarithmically in the cornea up to 6 hours post-infection. SLE scores increased from 0 to 30 hours post-infection for strains ATCC 14041 and 93-1399-1, while the SLE score of strain 94-EI-185-2 reached its maximum at 18 hours post-infection. Strain-specific differences in pathology were noted from 18 to 30 hours post-infection. Strain 94-EI-185-2 produced iritis but only mild corneal changes. Strain 93-1399-1 produced a severe corneal infiltrate encompassing the entire corneal surface as well as severe conjunctival inflammation and iritis. Strain ATCC 14041 produced a localized, severe, exudative corneal abscess that contained infecting bacteria. CONCLUSIONS: A rabbit model of Serratia keratitis was developed in which bacterial growth kinetics and strain-specific ocular pathologic changes were reproducible.

Effectiveness of ciprofloxacin-polystyrene sulfonate (PSS), ciprofloxacin and ofloxacin in a Staphylococcus keratitis model.

PURPOSE: Staphylococcus aureus causes severe corneal infections that often result in corneal scarring and blindness. Presently, therapy often involves the use of a fluoroquinolone antibiotic. This study, employing an experimental rabbit model of Staphylococcus keratitis, compared the effectiveness of two commonly prescribed formulations of fluoroquinolones to an experimental formulation, ciprofloxacin with polystyrene sulfonate (ciprofloxacin-PSS). The ciprofloxacin-PSS formulation uses an ion exchange resin to aid in the delivery of drug to the cornea. METHODS: Early (4-9 h postinfection, PI) and late (10-15 h PI) therapies were studied, employing 5 groups: ciprofloxacin-PSS, ciprofloxacin, ofloxacin, PSS vehicle. and untreated. Dosing regimens were: every 30 min, 60 min, or a single drop applied at 9 h PI. Eyes were observed by slit lamp examination (SLE) and bacterial colony forming units (CFU) per cornea were determined. RESULTS: Early phase therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min were equally effective (P > or = 0.2880), decreasing CFU per cornea by >5 log. Ciprofloxacin was significantly more active than ciprofloxacin-PSS or ofloxacin (P < or = 0.0410) when applied as a single drop. Late therapy with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin administered every 30 or 60 min resulted in >3 log decrease in CFU per cornea relative to controls (P < or = 0.0001). CONCLUSIONS: Topical treatment of experimental Staphylococcus keratitis with ciprofloxacin-PSS, ciprofloxacin, or ofloxacin was effective. The effectiveness of ciprofloxacin-PSS suggests that improved drug delivery systems employing an ion exchange resin could be useful in an ocular fluoroquinolone formulation.

Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa.

Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection. We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease. The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence. Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45 degreesC. Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen. Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors. The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease. Inhibition by dithiothreitol and beta-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity. The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection.

Neurophysiological function in farm workers exposed to organophosphate pesticides.

To investigate neurophysiological effects of low-level exposure to foliar organophosphate residues during one season among agricultural workers, the authors performed a cross-sectional study of 67 Hispanic farm workers and 68 age-, gender-, ethnicity-, and education-matched reference subjects. The neurophysiological examination included sensory and motor nerve conduction and neuromuscular junction testing. Erythrocyte cholinesterase activity was measured at the time of examination. No statistically significant neurophysiological differences between the exposed and reference groups were observed. Farm workers and reference subjects had similar sensory nerve latency and amplitude (sural), motor nerve conduction velocity (ulnar), and neuromuscular junction function (ulnar). No relationship between duration of exposure during the season and electrophysiological measures of nerve function was found. Exposure of farm workers to the low levels of organophosphate pesticides during one season experienced by farm workers in this study was not associated with impaired peripheral neurophysiological function.

Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.

Pseudomonas deficient in protease IV has significantly reduced corneal virulence.

PURPOSE: The role of protease IV in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated by comparing a mutant strain completely deficient in protease IV activity with its protease IV activity-producing parent. METHODS: A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which produces protease IV, through transposon insertion. Protease IV activity was determined by a casein agar assay, zymography, and cleavage of the chromogenic substrate, Chromozym PL. Corneal virulence was evaluated by slit lamp examination and bacterial cultures in both a rabbit intrastromal model and a mouse topical model of keratitis. RESULTS: The protease IV-deficient strain PA103-29::Tn9 had significantly reduced corneal virulence relative to its parent strain PA103-29 in both a rabbit intrastromal model and a mouse topical model of infection. In the rabbit model, ocular damage (slit lamp examination score) mediated by the parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infection. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model, eyes inoculated with the protease IV-producing parent strain had significant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant corneal damage during 6 days of infection. CONCLUSIONS: The ability to produce active protease IV was the determining factor in the severity of corneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications designed to minimize corneal damage during Pseudomonas keratitis.

Specific roles of alpha-toxin and beta-toxin during Staphylococcus aureus corneal infection.

Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha- and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha- or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr-, Hlb-, or Hla- strains. Epithelial erosions produced by parent strains were not produced by Agr- or Hla- strains. Hlb+ strains, unlike Hlb- strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla-), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb-). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.