SDG indicator 3.b.3 - an analysis of its robustness and challenges for measuring access to medicines for children.

BACKGROUND: Sustainable Development Goal (SDG) indicator 3.b.3 monitors progress in medicines' accessibility for adults and has significant limitations when applying to medicines for children. An adapted indicator methodology was developed to fill this gap, but no proof of its robustness exists. We provide this evidence through sensitivity analyses. METHODS: Data on availability and prices of child medicines from ten historical datasets were combined to create datasets for analysis: Dataset 1 (medicines selected at random) and Dataset 2 (preference given to available medicines, to better capture affordability of medicines). A base case scenario and univariate sensitivity analyses were performed to test critical components of the methodology, including the new variable of number of units needed for treatment (NUNT), disease burden (DB) weighting, and the National Poverty Line (NPL) limits. Additional analyses were run on a continuously smaller basket of medicines to explore the minimum number of medicines required. Mean facility scores for access were calculated and compared. RESULTS: The mean facility score for Dataset 1 and Dataset 2 within the base case scenario was 35.5% (range 8.0-58.8%) and 76.3% (range 57.2-90.6%). Different NUNT scenarios led to limited variations in mean facility scores of + 0.1% and -0.2%, or differences of + 4.4% and -2.1% at the more critical NPL of $5.50 (Dataset 1). For Dataset 2, variations to the NUNT generated differences of + 0.0% and -0.6%, at an NPL of $5.50 the differences were + 5.0 and -2.0%. Different approaches for weighting for DB induced considerable fluctuations of 9.0% and 11.2% respectively. Stable outcomes with less than 5% change in mean facility score were observed for a medicine basket down to 12 medicines. For smaller baskets, scores increased more rapidly with a widening range. CONCLUSION: This study has confirmed that the proposed adaptations to make SDG indicator 3.b.3 appropriate for children are robust, indicating that they could be an important addition to the official Global Indicator Framework. At least 12 child-appropriate medicines should be surveyed to obtain meaningful outcomes. General concerns that remain about the weighting of medicines for DB and the NPL should be considered at the 2025 planned review of this framework.

Comparison of mixed effects models of antimicrobial resistance metrics of livestock and poultry Salmonella isolates from a national monitoring system.

Antimicrobial use in agriculture is considered a pathway for the selection and dissemination of resistance determinants among animal and human populations. From 1997 through 2003 the U.S. National Antimicrobial Resistance Monitoring System (NARMS) tested clinical Salmonella isolates from multiple animal and environmental sources throughout the United States for resistance to panels of 16-19 antimicrobials. In this study we applied two mixed effects models, the generalized linear mixed model (GLMM) and accelerated failure time frailty (AFT-frailty) model, to susceptible/resistant and interval-censored minimum inhibitory concentration (MIC) metrics, respectively, from Salmonella enterica subspecies enterica serovar Typhimurium isolates from livestock and poultry. Objectives were to compare characteristics of the two models and to examine the effects of time, species, and multidrug resistance (MDR) on the resistance of isolates to individual antimicrobials, as revealed by the models. Fixed effects were year of sample collection, isolate source species and MDR indicators; laboratory study site was included as a random effect. MDR indicators were significant for every antimicrobial and were dominant effects in multivariable models. Temporal trends and source species influences varied by antimicrobial. In GLMMs, the intra-class correlation coefficient ranged up to 0.8, indicating that the proportion of variance accounted for by laboratory study site could be high. AFT models tended to be more sensitive, detecting more curvilinear temporal trends and species differences; however, high levels of left- or right-censoring made some models unstable and results uninterpretable. Results from GLMMs may be biased by cutoff criteria used to collapse MIC data into binary categories, and may miss signaling important trends or shifts if the series of antibiotic dilutions tested does not span a resistance threshold. Our findings demonstrate the challenges of measuring the AMR ecosystem and the complexity of interacting factors, and have implications for future monitoring. We include suggestions for future data collection and analyses, including alternative modeling approaches.

Pharmacological ascorbate with gemcitabine for the control of metastatic and node-positive pancreatic cancer (PACMAN): results from a phase I clinical trial.

BACKGROUND: Treatment for pancreatic cancer with pharmacological ascorbate (ascorbic acid, vitamin C) decreases tumor progression in preclinical models. A phase I clinical trial was performed to establish safety and tolerability of pharmacological ascorbate combined with gemcitabine in patients with biopsy-proven stage IV pancreatic adenocarcinoma. DESIGN: Nine subjects received twice-weekly intravenous ascorbate (15-125 g) employing Simon's accelerated titration design to achieve a targeted post-infusion plasma level of ≥350 mg/dL (≥20 mM). Subjects received concurrent gemcitabine. Disease burden, weight, performance status, hematologic and metabolic laboratories, time to progression and overall survival were monitored. RESULTS: Mean plasma ascorbate trough levels were significantly higher than baseline (1.46 ± 0.02 vs. 0.78 ± 0.09 mg/dL, i.e., 83 vs. 44 µM, p < 0.001). Adverse events attributable to the drug combination were rare and included diarrhea (n = 4) and dry mouth (n = 6). Dose-limiting criteria were not met for this study. Mean survival of subjects completing at least two cycles (8 weeks) of therapy was 13 ± 2 months. CONCLUSIONS: Data suggest pharmacologic ascorbate administered concurrently with gemcitabine is well tolerated. Initial data from this small sampling suggest some efficacy. Further studies powered to determine efficacy should be conducted.

Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis infection in United States dairy herds in 2007.

Testing of composite fecal (environmental) samples from high traffic areas in dairy herds has been shown to be a cost-effective and sensitive method for classification of herd status for Mycobacterium avium subsp. paratuberculosis (MAP). In the National Animal Health Monitoring System's (NAHMS) Dairy 2007 study, the apparent herd-level prevalence of MAP was 70.4% (369/524 had ≥ 1 culture-positive composite fecal samples out of 6 tested). Based on these data, the true herd-level prevalence (HP) of MAP infection was estimated using Bayesian methods adjusting for the herd sensitivity (HSe) and herd specificity (HSp) of the test method. The Bayesian prior for HSe of composite fecal cultures was based on data from the NAHMS Dairy 2002 study and the prior for HSp was based on expert opinion. The posterior median HP (base model) was 91.1% (95% probability interval, 81.6 to 99.3%) and estimates were most sensitive to the prior for HSe. The HP was higher than estimated from the NAHMS Dairy 1996 and 2002 studies but estimates are not directly comparable with those of prior NAHMS studies because of the different testing methods and criteria used for herd classification.

Inhibitors of hydroperoxide metabolism enhance ascorbate-induced cytotoxicity.

Pharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H(2)O(2) to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H(2)O(2). The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H(2)O(2) were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H(2)O(2) and depleted intracellular glutathione. When inhibitors of H(2)O(2) metabolism were combined with pharmacological ascorbate the increase in intracellular H(2)O(2) was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity.

Comparison of individual, pooled, and composite fecal sampling methods for detection of Salmonella on U.S. dairy operations.

The objectives of this study were to estimate the prevalence of Salmonella for individual, pooled, and composite fecal samples and to compare culture results from each sample type for determining herd Salmonella infection status and identifying Salmonella serovar(s). During the U.S. Department of Agriculture National Animal Health Monitoring System Dairy 2007 study, data and samples were collected from dairy operations in 17 major dairy states. As part of the study, composite fecal samples (six per operation) were collected from cow areas, such as holding pens, alleyways, and lagoons, where manure accumulates. Fecal samples also were collected from individual cows (35 per operation), and fecal sample pools were created by combining samples from 5 cows (7 per operation). A total of 1,541 composite fecal samples were collected from 260 operations in 17 states, and 406 (26.3%) of these samples were culture positive for Salmonella. Among the 116 operations for which all three sample types were obtained, 41.4% (48 operations) were Salmonella culture positive based on individual samples, 39.7% (46 operations) were positive based on pooled samples, and 49.1% (57 operations) were positive based on composite fecal samples. Relative to individual samples, the sensitivity of composite fecal samples for determining herd infection status was 85.4% and the sensitivity of pooled fecal samples was 91.7%. On 33.6% of operations (39 of 116), Salmonella was cultured from all three fecal sample types (individual, pooled, and composite), and 20 (51.3%) of these operations had exactly the same serovar in all three sample types. Use of composite fecal samples is less costly and time-consuming than use of individual or pooled samples and provides similar results for detecting the presence and identifying serovars of Salmonella in dairy herds. Therefore, composite sampling may be an appropriate alternative to culture of individual samples when assessing Salmonella status in dairy herds.

Clostridium difficile from healthy food animals: optimized isolation and prevalence.

Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by alcohol shock and isolation on tryptic soy agar supplemented with 5% sheep blood, and cycloserine-cefoxitin fructose agar. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by isolation on tryptic soy agar supplemented with 5% sheep blood and cycloserine-cefoxitin fructose agar. A total of 55 (15.9%, n = 345) swine fecal samples, 32 (2.4%, n = 1,325) dairy cattle fecal samples, and 188 (6.3%, n = 2,965) beef cattle fecal samples were positive for C. difficile by either method. However, the DS was significantly better than the SS for the recovery of C. difficile from swine feces, while the SS was significantly better than the DS for the recovery of C. difficile from beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study suggests that food animals might harbor C. difficile and it provides critical information that isolation methods might not have universal application across animal species.

Risk factors associated with the presence of viable Listeria monocytogenes in bulk tank milk from US dairies.

The objective of the study was to screen a large number of herd management practices and herd characteristics from US dairies to identify herd-level risk factors associated with the presence of Listeria monocytogenes in bulk tank milk (BTM). A total of 71 variables was univariately evaluated for their association with the presence of L. monocytogenes in BTM. Results from the univariate analysis indicated that using automatic take offs and having an open herd management increased the risk of BTM contamination with L. monocytogenes, while storing manure in outside pens not accessible to cattle decreased the risk. These variables, however, were not sustained in the multivariable model, which indicated that the presence of L. monocytogenes in BTM was significantly associated with region of the country (farms in the southeast and northeast were six and four times more likely respectively, to have BTM contamination than farms in the west) and number of milking cows (herds with >500 milking cows were five times more likely to have BTM contamination than herds with <100 milking cows). In conclusion, our results suggest that risk factors associated with BTM contamination are different depending on the geographical region and herd size of the operation.

Risk associated with transportation and lairage on hide contamination with Salmonella enterica in finished beef cattle at slaughter.

Transportation of cattle to the slaughter plant could influence hide contamination with Salmonella enterica. Fecal and hide samples were obtained from 40 lots of cattle at the feedlot and again at the slaughter plant. Potential risk factors for hide contamination were evaluated. A multilevel Poisson regression model was used to determine whether transportation and lairage were associated with hide contamination by Salmonella. Cattle with hide samples positive for Salmonella at the feedlot had twice the risk of having positive slaughter hide samples compared with cattle without positive feedlot hide samples (relative risk [RR], 1.9). Cattle transported in trailers from which samples positive for Salmonella were collected had twice the risk of having positive slaughter hide samples compared with cattle transported in culture-negative trailers (RR, 2.3). Cattle transported for long distances had twice the risk of having positive hide samples at slaughter compared with cattle transported shorter distances (RR, 2.3). Cattle held in lairage pens contaminated with feces had twice the risk of having positive slaughter hide samples compared with cattle held in clean pens (RR, 1.8). Cattle held off feed longer than 18 h before loading had twice the risk of having positive slaughter hide samples compared with cattle held off feed for shorter times (RR, 1.7). Cattle that were agitated during loading had twice the risk of having positive slaughter hide samples compared with cattle that were calm (RR, 2.2). These findings suggest that variables associated with transportation and lairage can impact the presence of Salmonella on the hides of cattle at slaughter.

Impact of transportation and lairage on hide contamination with Escherichia coli O157 in finished beef cattle.

Transportation of cattle from the feedlot to the slaughter plant could influence hide contamination of Escherichia coli O157. A study was initiated to investigate the influence of transportation and lairage on shedding and hide contamination of E. coli O157. Fecal and hide samples were obtained from 40 pens of harvest-ready beef cattle at the feedlot prior to transport and again at the slaughter plant immediately after slaughter. Potential risk factors for hide contamination at the feedlot, during transport, and at slaughter were evaluated. A multilevel Poisson regression model was used to evaluate if transportation and lairage were associated with hide contamination by E. coli O157 in finished beef cattle. Lots of cattle held in E. coli O157-positive lairage pens had eight times greater risk of having positive slaughter hide samples compared with cattle held in culture-negative pens (relative risk, 8.0; 95% confidence interval, 1.6 to 38.8). Lots of cattle that were held in lairage pens contaminated with feces had three times greater risk for positive slaughter hide samples compared with cattle held in clean pens (relative risk, 3.1; 95% confidence interval, 1.2 to 7.9). Lots of cattle that were transported for long distances (> 160.9 km) had twice the risk of having positive hide samples at slaughter compared with cattle transported a shorter distance (relative risk, 2.4; 95% confidence interval, 1.1 to 5.1). These findings suggest that transportation and lairage should be considered in E. coli O157 control strategies.

Factors associated with occurrence and recovery of nonambulatory dairy cows in the United States.

The primary objective of this study was to compare characteristics of US dairy operations that had one or more nonambulatory cows (unable to rise for any period of time) (cases) with operations that had no nonambulatory cows (controls) during 2004. A secondary objective was to describe factors associated with recovery of the last nonambulatory cow on the operation during 2004. Case dairy operations (n = 1,822) more often fed a total mixed ration [odds ratio (OR) = 2.0; confidence interval (CI): 1.1-3.4], produced more than 9,090 kg of milk (OR = 2.8; CI: 1.8-4.5), and were more likely to be of medium to large herd size (100 or more head of adult cows, OR = 3.7; CI: 2.2-6.2) compared with control dairies (n = 151). Compared with operations where the predominant flooring surface on which lactating cows stood or walked in winter was pasture, operations where pasture was not the predominant surface were at increased risk of having nonambulatory cows (OR = 4.7; CI: 2.2-10.2). Cows nonambulatory for less than 24 h were more likely to recover compared with cows nonambulatory for 24 h or more (OR = 3.0; CI: 2.0-4.4). Cows that received calcium, phosphorus, or potassium while non-ambulatory were more likely to recover (OR = 3.6; CI: 2.1-6.1) than cattle that did not receive these treatments. Cattle that were not repositioned periodically were more likely to recover (OR = 2.1; CI: 1.4-3.1), as were cattle that were not treated by a veterinarian before becoming nonambulatory (OR = 1.9; CI: 1.1-3.3). These findings are consistent with prolonged recumbency and prior history of health issues, respectively. Nonambulatory cattle with hypocalcemia were more likely to recover (OR = 6.0; CI: 3.4-10.7) compared with nonambulatory cows with all other causes of a nonambulatory condition (analyzed collectively as a single variable but including cancer, clinical mastitis, digestive conditions, metabolic imbalances, neurological problems, respiratory disease, other, unknown). The results of this study reveal that the majority of US dairy operations have at least one nonambulatory dairy cow over the course of a year. Additionally, individual animal factors associated with being nonambulatory may lead to improved identification and treatment of animals that are nonambulatory for a prolonged period. From the perspective of recovery, considering euthanasia is appropriate for cows that have been nonambulatory for more than 24 h.

Risk factors for owner-reported occurrence of equine protozoal myeloencephalitis in the US equine population.

BACKGROUND: Equine protozoal myeloencephalitis (EPM) is a serious and often fatal neurologic disease of horses, but few studies have investigated risk factors. OBJECTIVES: To evaluate operation- and individual-level factors associated with likelihood of the occurrence of EPM. ANIMALS: Data were collected as part of a study of the US equine industry from 1,178 operations representing 83.9% of horses and 51.6% of operations with > or =3 horses in 28 states. METHODS: Probability-based sampling was used to enroll representative operations in a cross-sectional study. Interviews were conducted to collect information regarding health and management of horses. A nested case-control study was used to investigate risk factors among individual horses. Interview data were combined with climate data, human population density, and opossum regional ecology categories. Data were analyzed using logistic regression to identify risk factors for the occurrence of EPM. RESULTS: Owners reported that 95% of EPM cases included in this study were diagnosed by veterinarians. Variables associated with EPM occurrence on premises included opossum regional ecology, reported exposure to small wildlife, climate, terrain, housing, choice of bedding material, method of storing feeds, equine stocking density, and primary use of horses. Among individual horses, age was most strongly associated with disease risk. Associations also were identified with sex, breed, primary use, and participation in competitions. CONCLUSIONS AND CLINICAL IMPORTANCE: Because the risk of EPM occurrence on operations is closely tied to factors that impact exposure to opossums, their feces, and their environment, controlling these exposures may be important in preventing the occurrence of EPM.

Evaluation of factors associated with increased dairy cow mortality on United States dairy operations.

Dairy cow mortality is an increasingly severe problem for the US dairy industry. The objective of this study was to examine a variety of herd management practices and herd characteristics to identify factors associated with increased cow mortality in US dairy herds. The National Animal Health Monitoring System's Dairy 2002 study surveyed dairy operations in 21 major dairy states. The complete data set included results from 953 dairy farms with a minimum of 30 dairy cows per farm. Associations between dairy cow mortality and 119 a priori-selected management practices or characteristics of 953 operations were evaluated. Eighty of the 119 risk factors explored in a univariate analysis met initial inclusion criteria for further evaluation of association with dairy cow mortality. A multivariable analysis was conducted to explore more complex relationships. The final multivariable model included 7 representative variables: herd levels of respiratory disease, lameness, and antibiotic use for treating sick cows, the percentage of culled cows less than 50 d in milk, the average calving interval, the use of a total mixed ration, and the region of the country. Increased odds of a greater level of mortality on farms was associated with greater percentages of lameness, respiratory disease, and sick cows treated with antibiotics, demonstrating the influence of physical derangements and disease on dairy cow mortality. Increased odds of a greater level of mortality was also associated with feeding a total mixed ration, culling fewer cows in early lactation, and herds located in western, midwestern, and southeastern regions relative to the northeastern United States, pointing to the importance of management decisions and operation characteristics on mortality outcomes. Further, an important interplay between facets of health and management on dairy cow mortality was suggested through the inclusion of the calving interval, with a longer calving interval leading to increased odds of a greater level of mortality on farms. Analysis of a variety of herd characteristics and practices with nationally representative data suggests that several health problems in tandem with aspects of operational construct and management are associated with increasing mortality.

Herd management practices and their association with bulk tank somatic cell count on United States dairy operations.

The objective of this study was to evaluate associations between bulk tank somatic cell count (BTSCC) and herd management practices using data collected in the National Animal Health Monitoring System Dairy 2002 study. Twenty-six percent and 17.8% of 1,013 operations reported a BTSCC < 200,000 cells/mL and > 400,000 cells/mL, respectively. Univariate analysis identified associations between management variables and BTSCC. The use of mattresses, sand, and newspaper as bedding were all associated with a lower BTSCC. Primary lactating cow housing facility, outside maternity housing area, flooring type cows walk or stand on, and use of automatic take-offs were also associated with BTSCC. Multivariate associations between management variables and BTSCC were determined by backward elimination ordinal logistic regression. The odds of an operation from the West, Midwest, and Northeast having a high BTSCC were lower than those from the Southeast. The odds of a higher BTSCC were 2 times greater for operations with a rolling herd average milk production < 9,090 kg/cow per year compared with those with > or = 9,090 kg/cow per year. Operations using composted manure were 2.9 times more likely to have a higher BTSCC than those not using composted manure. Finally, operations that reported not using a coliform mastitis vaccine were 1.7 times more likely to have a higher BTSCC than those using one. Future studies of the association between management practices and BTSCC should include an evaluation of the quality of management practice application and herd prevalence of contagious mastitis pathogens. Significant variables identified in this study dealt with housing, use of composted manure for bedding, and coliform mastitis vaccine use, suggesting the effect of environmental mastitis pathogens may be more influential on BTSCC than previously thought.

Evaluation of environmental sampling and culture to determine Mycobacterium avium subspecies paratuberculosis distribution and herd infection status on US dairy operations.

The objectives of this study were to determine the distribution of Mycobacterium avium subspecies paratuberculosis (MAP) in the environment and assess the relationship between the culture status of MAP in the farm environment and herd infection status. The National Animal Health Monitoring System's Dairy 2002 study surveyed dairy operations in 21 states. One component of the study involved collection and culturing of environmental samples for MAP from areas on farms where manure accumulated from a majority of a herd's cows. Operations were selected for inclusion based on perceived risk factors for MAP infection identified in a previously administered questionnaire. Individual animal and environmental samples were collected and used to determine the efficiency of environmental sampling for determination of herd infection status. Individual animal fecal, serum, and milk samples were used to classify herds as infected or not infected based on the presence of at least one test-positive animal in the herd. A total of 483 environmental samples (approximately 5 per farm) were collected, and 218 (45.1%) were culture-positive for MAP. A similar percentage of environmental cultures collected from all designated areas were positive [parlor exits (52.3%), floors of holding pens (49.1%), common alleyways (48.8%), lagoons (47.4%), manure spreaders (42.3%), and manure pits (41.5%)]. Of the 98 operations tested with the environmental sample culture, 97 had individual serum ELISA results, 60 had individual fecal culture results, and 34 had individual milk ELISA results. Sixty-nine of the 98 operations (70.4%) had at least one environmental sample that was culture-positive. Of the 50 herds classified as infected by fecal culture, 38 (76.0%) were identified by environmental culture. Two of the 10 operations classified as not infected based on individual animal fecal culture were environmental culture-positive. Of the 80 operations classified as infected based on serum ELISA-positive results, 61 (76.3%) were identified as environmental-positive, whereas 20 of the 28 (71.4%) operations identified as infected based on milk ELISA were detected by environmental sampling. Environmental sample culturing is less costly than individual animal sampling, does not require animal restraint, and identified more than 70% of infected operations. Environmental sampling is another diagnostic tool that veterinarians and dairy producers can use to determine herd infection status for MAP.

Effect of diagnostic testing error on intracluster correlation coefficient estimation.

Estimation of the intracluster correlation coefficient (ICC) for infectious animal diseases may be of interest for survey planning and for calculating variance inflation factors for estimators of prevalence. Typically, diagnostic tests with imperfect sensitivity and specificity are used in surveys. In such studies, where animals from multiple herds are tested, the ICC often is estimated using apparent (test-based) rather than true prevalence data. Through Monte Carlo simulation, we examined the effect of substituting diagnostic test outcomes for true infection status on an ANOVA estimator of ICC, which was designed for use with true infection status data. We considered effects of diagnostic test sensitivity and specificity on the estimated ICC when the true ICC value and infection status of the sampled individuals were known. The ANOVA estimator underestimated the true ICC when the diagnostic test was imperfect. We also demonstrated, under the beta-binomial model, that the ICC based on apparent infection status for individuals is < or = ICC based on true infection status. In addition, we propose a Bayesian model for estimating the ICC that incorporates imperfect sensitivity and specificity and illustrate the Bayesian model using a simulation study and one example; a seroprevalence survey of ovine progressive pneumonia in U.S. sheep flocks.

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.

Relationships between fecal culture, ELISA, and bulk tank milk test results for Johne's disease in US dairy herds.

Objectives were to estimate percentages of seropositive herds with cows shedding Mycobacterium paratuberculosis in feces and milk, and to estimate sensitivity, specificity, and predictive value of an ELISA relative to fecal culture. Dairy cows (n = 712) were randomly selected from 61 herds previously identified by ELISA as positive for Johne's disease. Fecal and bulk tank milk samples (n = 52 of 61 herds) were obtained from 10 states in the United States. Fecal samples were processed by a double centrifugation, double decontamination culture procedure. Milk samples were processed for both culture and DNA analysis by using polymerase chain reaction (PCR). Of 24 herds with at least three cows that had tested ELISA-positive, 79% were also culture-positive, compared with 18 of 37 herds with one or two ELISA-positive cows. Both fecal-culture and ELISA results were available on 651 cows; only 25% of cows that were fecal-culture positive also tested positive by ELISA and over 6% of cows that were fecal-culture negative tested ELISA-positive. Milk samples all cultured negative, but analysis of milk samples by PCR resulted in 68% of herds positive for M. paratuberculosis DNA including 24 of 31 herds with positive fecal cultures and 11 of 21 herds with negative fecal cultures. Sensitivity and specificity of the ELISA compared with fecal culture is lower than previously reported and perhaps best used in screening herds because of limited efficacy to predict infection in individual cows. In addition, contamination of bulk tank milk samples with M. paratuberculosis does occur in seropositive herds, even in some with negative fecal cultures.

Prevalence of antibodies against Mycobacterium avium subsp paratuberculosis among beef cow-calf herds.

OBJECTIVE: To estimate the prevalence of Mycobacterium avium subsp paratuberculosis infection among cows on beef operations in the United States. DESIGN: Cross-sectional seroprevalence study. Sample Population-A convenience sample of 380 herds in 21 states. PROCEDURES: Serum samples were obtained from 10,371 cows and tested for antibodies to M avium subsp paratuberculosis with a commercial ELISA. Producers were interviewed to collect data on herd management practices. RESULTS: 30 (7.9%) herds had 1 or more animals for which results of the ELISA were positive; 40 (0.4%) of the individual cow samples yielded positive results. None of the herd management practices studied were found to be associated with whether any animals in the herd would be positive for antibodies to M avium subsp paratuberculosis. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prevalence of antibodies to M avium subsp paratuberculosis among beef cows in the United States is low. Herds with seropositive animals were widely distributed geographically.