ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structures.

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show that the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly outnumber Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures.

Quantification of the sensitivity of early detection surveillance.

Early detection surveillance is used for various purposes, including the early detection of non-communicable diseases (e.g. cancer screening), of unusual increases of disease frequency (e.g. influenza or pertussis outbreaks), and the first occurrence of a disease in a previously free population. This latter purpose is particularly important due to the high consequences and cost of delayed detection of a disease moving to a new population. Quantifying the sensitivity of early detection surveillance allows important aspects of the performance of different systems, approaches and authorities to be evaluated, compared and improved. While quantitative evaluation of the sensitivity of other branches of surveillance has been available for many years, development has lagged in the area of early detection, arguably one of the most important purposes of surveillance. This paper, using mostly animal health examples, develops a simple approach to quantifying the sensitivity of early detection surveillance, in terms of population coverage, temporal coverage and detection sensitivity. This approach is extended to quantify the benefits of risk-based approaches to early detection surveillance. Population-based clinical surveillance (based on either farmers and their veterinarians, or patients and their local health services) provides the best combination of sensitivity, practicality and cost-effectiveness. These systems can be significantly enhanced by removing disincentives to reporting, for instance by implementing effective strategies to improve farmer awareness and engagement with health services and addressing the challenges of well-intentioned disease notification policies that inadvertently impose barriers to reporting.

Evaluation of testing strategies to identify infected animals at a single round of testing within dairy herds known to be infected with Mycobacterium avium ssp. paratuberculosis.

As part of a broader control strategy within herds known to be infected with Mycobacterium avium ssp. paratuberculosis (MAP), individual animal testing is generally conducted to identify infected animals for action, usually culling. Opportunities are now available to quantitatively compare different testing strategies (combinations of tests) in known infected herds. This study evaluates the effectiveness, cost, and cost-effectiveness of different testing strategies to identify infected animals at a single round of testing within dairy herds known to be MAP infected. A model was developed, taking account of both within-herd infection dynamics and test performance, to simulate the use of different tests at a single round of testing in a known infected herd. Model inputs included the number of animals at different stages of infection, the sensitivity and specificity of each test, and the costs of testing and culling. Testing strategies included either milk or serum ELISA alone or with fecal culture in series. Model outputs included effectiveness (detection fraction, the proportion of truly infected animals in the herd that are successfully detected by the testing strategy), cost, and cost-effectiveness (testing cost per true positive detected, total cost per true positive detected). Several assumptions were made: MAP was introduced with a single animal and no management interventions were implemented to limit within-herd transmission of MAP before this test. In medium herds, between 7 and 26% of infected animals are detected at a single round of testing, the former using the milk ELISA and fecal culture in series 5 yr after MAP introduction and the latter using fecal culture alone 15 yr after MAP introduction. The combined costs of testing and culling at a single round of testing increases with time since introduction of MAP infection, with culling costs being much greater than testing costs. The cost-effectiveness of testing varied by testing strategy. It was also greater at 5 yr, compared with 10 or 15 yr, since MAP introduction, highlighting the importance of early detection. Future work is needed to evaluate these testing strategies in subsequent rounds of testing as well as accounting for different herd dynamics and different levels of herd biocontainment.

The consequences of risk-based surveillance: Developing output-based standards for surveillance to demonstrate freedom from disease.

Output-based surveillance standards provide a mechanism to achieve harmonised and comparable surveillance (which meets a defined objective) while allowing flexible approaches that are adapted to the different populations under surveillance. When correctly implemented, they can result in lower cost and greater protection against disease spread. This paper presents examples of how risk-based sampling can improve the efficiency of surveillance, and describes the evolution of output-based surveillance standards for demonstration of freedom from disease in terms of three generations of approach: surveillance sensitivity, probability of freedom, and expected cost of error. These three approaches progressively capture more of the factors affecting the final outcome. The first two are relatively well accepted but the third is new and relates to the consequences of infection. There has been an increased recognition of the value of risk-based sampling for demonstration of freedom from disease over the last decades, but there has been some disagreement about practical definitions and implementation, in particular as to whether 'risk-based' implies probability of infection or probability and consequences. This paper argues that risk-based sampling should be based solely on the probability of infection of a unit within the population, while the consequences of infection should be used to set the target probability of freedom. This approach provides a quantitative framework for planning surveillance which is intuitively understandable. The best way to find disease, if it is present, is to focus on those units that are most likely to be infected. However, if the purpose of surveillance includes mitigating the risk of a disease outbreak, we want to ensure that that risk is smallest in those populations where the consequences of failure to detect are greatest.

Demonstrating freedom from disease using multiple complex data sources 2: case study--classical swine fever in Denmark.

A method for quantitative evaluation of surveillance for disease freedom has been presented in the accompanying paper (Martin et al., 2007). This paper presents an application of the methods, using as an example surveillance for classical swine fever (CSF) in Denmark in 2005. A scenario tree model is presented for the abattoir-based serology component of the Danish CSF surveillance system, in which blood samples are collected in an ad hoc abattoir sampling process, from adult pigs originating in breeding herds in Denmark. The model incorporates effects of targeting (differential risk of seropositivity) associated with age and location (county), and disease clustering within herds. A surveillance time period of one month was used in the analysis. Records for the year 2005 were analysed, representing 25,332 samples from 3528 herds; all were negative for CSF-specific antibodies. Design prevalences of 0.1-1% of herds and 5% of animals within an infected herd were used. The estimated mean surveillance system component (SSC) sensitivities (probability that the SSC would give a positive outcome given the animals processed and that the country is infected at the design prevalences) per month were 0.18, 0.63 and 0.86, for among-herd design prevalences of 0.001, 0.005 and 0.01. The probabilities that the population was free from CSF at each of these design prevalences, after a year of accumulated negative surveillance data, were 0.91, 1.00 and 1.00. Targeting adults and herds from South Jutland was estimated to give approximately 1.9, 1.6 and 1.4 times the surveillance sensitivity of a proportionally representative sampling program for these three among-herd design prevalences.

Demonstrating freedom from disease using multiple complex data sources 1: a new methodology based on scenario trees.

Current methods to demonstrate zone or country freedom from disease are based on either quantitative analysis of the results of structured representative surveys, or qualitative assessments of multiple sources of evidence (including complex non-representative sources). This paper presents a methodology for objective quantitative analysis of multiple complex data sources to support claims of freedom from disease. Stochastic scenario tree models are used to describe each component of a surveillance system (SSC), and used to estimate the sensitivity of each SSC. The process of building and analysing the models is described, as well as techniques to take into account any lack of independence between units at different levels within a SSC. The combination of sensitivity estimates from multiple SSCs into a single estimate for the entire surveillance system is also considered, again taking into account lack of independence between components. A sensitivity ratio is used to compare different components of a surveillance system. Finally, calculation of the probability of country freedom from the estimated sensitivity of the surveillance system is illustrated, incorporating the use and valuation of historical surveillance evidence.

Induction of parturition in dairy cattle and its effects on health and subsequent lactation and reproductive performance.

OBJECTIVE: To determine the effect of induction of parturition on health, milk production and reproductive performance of dairy cows. DESIGN: A prospective cohort study in 62 dairy herds. PROCEDURE: Health, milk production and fertility indices were documented for 1449 dairy cows treated with dexamethasone trimethylacetate, with or without prostaglandin to induce calving. Equivalent data was collected for 603 untreated herd mates that calved at approximately the same time. RESULTS: The median interval from initial treatment to calving was 11 days. Induction was associated with a substantially lower calf survival and commercial value of surviving calves. Calf viability and value was lower when induced cows were at an earlier stage of pregnancy. Retained foetal membranes, photosensitisation and other problems were significantly more frequent in the induced group compared to the untreated group. Milk production of induced cows was approximately 4% lower than untreated ones, but the majority of reproductive indices were not significantly different between the two groups. CONCLUSIONS: The practice of induction of parturition in seasonal calving dairy herds is a reliable way of shortening the gestation period of cows. Costs associated with morbidity and mortality of induced cows and losses in lactation and calf production are offset by benefits of improved reproductive performance and more efficient management of the herd. The welfare aspects of induction on calf survival must be considered.

Data management and analysis systems for bluetongue virus zoning in Australia.

Bluetongue virus (BTV) monitoring data in Australia is managed using a secure web-enabled centralised database. Scientists across the country submit virological and entomological data from sentinel and other sites using the Internet. Automated reporting and mapping systems make this data immediately available to all users. This system underpins the process used for defining zone boundaries. Immediate access to monitoring results allows the zones to be redefined as soon as any evidence of expansion of the area of BTV activity is detected. The method used to define zone boundaries, incorporating detailed information on vector and virus activity, property boundaries and subdivision boundaries, geography and climate, is described.

The impact of current and proposed changes to general guidelines on bluetongue surveillance of the Office International des Epizooties.

New draft guidelines for surveillance have been prepared for possible submission to the Office International des Epizooties (OIE) General Session for adoption in 2005. These guidelines are non-prescriptive and output-oriented, but also identify a series of critical elements required to effectively implement and evaluate surveillance systems. The guidelines allow for the use of a range of approaches to surveillance, including the use of non-random data sources and the combination of multiple sources of evidence. They also require transparency and the presence of basic quality assurance systems. The guidelines deal with surveillance to demonstrate freedom from infection as well as surveillance to determine the distribution and occurrence of infection. If the draft guidelines are adopted, a range of novel approaches to surveillance of bluetongue virus (BTV) may become formally acknowledged and accepted under the OIE Terrestrial animal health code. This may enable different countries to tailor their BTV surveillance systems more closely to their own needs and capabilities while maintaining equivalence in the outputs of the systems.

Spatial expression of Hox cluster genes in the ontogeny of a sea urchin.

The Hox cluster of the sea urchin Strongylocentrous purpuratus contains ten genes in a 500 kb span of the genome. Only two of these genes are expressed during embryogenesis, while all of eight genes tested are expressed during development of the adult body plan in the larval stage. We report the spatial expression during larval development of the five 'posterior' genes of the cluster: SpHox7, SpHox8, SpHox9/10, SpHox11/13a and SpHox11/13b. The five genes exhibit a dynamic, largely mesodermal program of expression. Only SpHox7 displays extensive expression within the pentameral rudiment itself. A spatially sequential and colinear arrangement of expression domains is found in the somatocoels, the paired posterior mesodermal structures that will become the adult perivisceral coeloms. No such sequential expression pattern is observed in endodermal, epidermal or neural tissues of either the larva or the presumptive juvenile sea urchin. The spatial expression patterns of the Hox genes illuminate the evolutionary process by which the pentameral echinoderm body plan emerged from a bilateral ancestor.

A new probability formula for surveys to substantiate freedom from disease.

Surveys to substantiate freedom from disease are becoming increasingly important. This is due to the changes in rules governing international trade in animals and animal products, and to an increase in disease eradication and herd-level accreditation schemes. To provide the necessary assurances, these surveys must have a sound theoretical basis. Until now, most surveys have been based on the assumption that the screening test used was perfect (sensitivity and specificity both equal to one), and/or that the study population was infinite. Clearly, these assumptions are virtually always invalid. This paper presents a new formula that calculates the exact probability of detecting diseased animals, and considers both imperfect tests and finite population size. This formula is computationally inconvenient, and an approximation that is simpler to calculate is also presented. The use of these formulae for sample-size calculation and analysis of survey results is discussed. A computer program, 'FreeCalc', implementing the formulae is presented along with examples of sample size calculation for two different scenarios. These formulae and computer program enable the accurate calculation of survey sample-size requirements, and the precise analysis of survey results. As a result, survey costs can be minimised, and survey results will reliably provide the required level of proof.

Two-stage sampling in surveys to substantiate freedom from disease.

Disease in livestock populations tends to cluster at the herd level. In order to account for this--and to overcome the problems of simple random sampling from a very large population--large-scale livestock surveys usually involve two-stage sampling. However, the use of two-stage sampling presents particular problems for sample-size calculation and analysis. We developed a probability formula for two-stage sampling, initially based on the assumption of a perfect test. We used this formula to demonstrate how combinations of first-stage (number of herds) and second-stage (number of animals in selected herds) sample sizes can be altered to achieve a least-cost survey, and used simulation to validate the formula. To overcome the unrealistic assumption of a perfect test, we then applied an exact-probability formula (which takes imperfect tests and finite population sizes into account) to the two-stage sampling design. An example is given which shows how implementing the formula with the FreeCalc computer program allows least-cost first and second-stage sample sizes to be calculated.

Implementation of internal laboratory quality control procedures for the monitoring of ELISA performance at a regional veterinary laboratory.

Quality control (QC) procedures for antigen detection enzyme-linked immunosorbent assays (ELISAs) for hog cholera (HC) virus, foot and mouth disease (FMD) virus, and an antibody detection ELISA for FMD virus were established at a regional veterinary laboratory in northern Thailand. A recently developed computer software package, QCEL, was used to facilitate management and analysis of QC data. The program was used to assess test performance by producing Shewhart-CUSUM control charts which monitored control data for unacceptable fluctuations or trends. QCEL-generated control charts and analyses are presented and discussed. The use of a simple integrated computerised system for storage and analysis of QC control data provided the laboratory with the opportunity to achieve increased confidence in the results of tests performed.

Evaluation of an ultrasonic Doppler probe for pregnancy diagnosis in cattle.

The use of an ultrasonic Doppler probe for pregnancy diagnosis was evaluated. Data from the examination of 1115 cattle were analysed. Comparison of the probe with manual rectal palpation by an experienced clinician yielded a sensitivity of 92.8 +/- 1.6% (+/- 95% confidence interval) and a specificity of 75.8 +/- 7.4%. This level of accuracy was found to be insufficient to recommend the probe be used by farmers for the diagnosis of pregnancy.

A survey of the efficacy of sustained-release monensin capsules in the control of bloat in dairy cattle.

Thirty-four farms in the Macalister Irrigation District in Gippsland, Victoria, using sustained-release monensin capsules in 5102 cattle in the 1990 bloat season were surveyed. Questions were asked about the prevention and incidence of bloat in 1989 and 1990. Eight farms not using the capsules were selected to act as controls. Relatively low rates of broken capsules (0.6%), injury to animals at administration (0.06%) and regurgitation (1.02%) were reported. A variety of preventive techniques were used. There was a significant decrease in the use of pasture spraying, drinking water administration and flank spraying of anti-bloat substances on the farms using the capsules in the 1990 season, with no compensatory rise in the use of other techniques. There was no significant change in bloat prevention techniques used on the control farms over the same period. Significantly fewer cattle on the farms using capsules were treated for, and fewer died of clinical bloat in 1990 than in 1989. There was no significant change in the incidence of bloat on the control farms over the same period. It was considered that the anti-bloat capsules were effective in reducing the incidence of clinical bloat in pasture-fed dairy cattle.

Relationship between milk production and somatic cell count in dairy cows in East Gippsland.

The analysis of records for 75,821 herd tests on 23,700 cows in the Macalister Irrigation District of East Gippsland, using a model that also included stage of lactation, herd and cow effects, found a strong relationship between somatic cell count and milk production. Unit increases in the natural logarithm of the somatic cell count were associated with decreases in production of up to 2.6% in the range 54,598 to 403,429 cells per mL. This is equivalent to a decrease of 1.9% for a doubling of cell count from 100,000 to 200,000 cells per mL. Subclinical mastitis was assessed to be a significant source of loss in milk production in the study area.

Electrical and anatomic characteristics of cells of ferret paratracheal ganglion.

A preparation has been developed for study of the electrical properties of the neurons of the ferret paratracheal ganglia. Two cell types were identified. AH cells were characterized by the presence of a single action potential in response to cathodal current pulses, followed by a profound after hyperpolarization that lasted for several hundred milliseconds. Electrical stimulation of branches of the laryngeal nerves produced a fast excitatory postsynaptic potential (EPSP) that was often followed by an apparent inhibitory postsynaptic potential (IPSP). A similar response was seen with electrical stimulation of interganglionic nerve trunks. Hexamethonium reversibly inhibited fast EPSPs in these cells. Type B cells could not be excited by intracellular current injections. At least some of these cells appear to be neurons as electrical stimulation of nerve trunks resulted in a slow EPSP and, rarely, a fast IPSP. Ganglia have between 10 and 20 cell bodies, and their diameters are between 15 and 40 microns. Two types of nerve endings were seen: 1) those with mainly round agranular vesicles 50-60 nm in diameter, and 2) those with large dense-cored vesicles of approximately 100 nm with an electron-lucent halo around the core.

The quest for the inhibitory neurotransmitter in bovine tracheal smooth muscle.

The effect of inhibitory nerve stimulation on the mechanical, membrane potential and membrane conductance responses of isolated bovine tracheal smooth muscle has been studied. Membrane responses were measured in a sucrose-gap apparatus. In order to record inhibitory responses, it was necessary to increase tone in the preparation by applying a drug such as histamine. When tone was raised, repetitive field stimulation of intrinsic nerves caused depolarization and contraction, followed by relaxation and a suppression of histamine-induced slow waves. Hyperpolarization of the membrane was only seen following prolonged nerve stimulation, and there was no change in membrane conductance. The inhibitory effect of nerve stimulation was abolished by tetrodotoxin, but was not abolished by atropine, indomethacin, propranolol, naloxone or the purinergic blockers quinidine and theophylline. It was not satisfactorily mimicked by catecholamines, by gamma-amino-n-butyric acid (GABA) or by purines. Nerves with catecholamine fluorescence could not be found in the tracheal muscle layer. Neither adrenergic nor purinergic types of nerve terminal could be found in the tracheal muscle layer during ultrastructural examination of over one thousand nerve profiles. Vasoactive intestinal peptide (VIP) caused relaxation of the histamine-contracted tracheal muscle, suppressed the slow wave and caused slight hyperpolarization at higher concentrations, without affecting the membrane conductance. VIP was found in samples of tracheal muscle at a mean concentration of 1.95 ng/g. When the effluent solution flowing past isolated tracheal muscle strips was assayed for VIP, samples collected during inhibitory nerve stimulation had much higher concentrations of the peptide than samples collected before stimulation, after stimulation, or during stimulation in the presence of tetrodotoxin (10(-6) mol/l). The VIP content of the effluent during control periods was 73.8 pg/ml, and during stimulation was 167.5 pg/ml. It is suggested that VIP might be the non-adrenergic inhibitory neurotransmitter in bovine tracheal smooth muscle.

Depolarization and increased conductance precede superoxide release by concanavalin A-stimulated rat alveolar macrophages.

Rat alveolar macrophages release superoxide into the extracellular medium when stimulated by concanavalin A. This process, the respiratory burst, is characterized by a delay between binding of the stimulus and release of superoxide. It has been proposed that a key event that occurs during this delay period is the alteration of membrane electrical potential. Microelectrode impalement was used to directly measure electrical properties of the plasma membrane. Upon addition of concanavalin A, the membrane potential depolarized 21%, and membrane electrical resistance decreased 16%. Parallel chemical measurement of superoxide release indicated that these changes in electrical properties precede the release of superoxide.