We evaluated the effect of 4 anthelmintic treatments on the viability of Trichinella spiralis encysted muscle larvae (ML) 55 days post infection (PI) in experimentally infected pigs. Muscle larvae were isolated from pig muscle by artificial digestion after oral treatment of pigs with Levamisole (8mg/kg, daily for 5 days) and Mebendazole (50mg/kg, daily for 5 days); Doramectin (0.3mg/kg, single IM injection), and Moxidectin (0.5mg/kg, single pour on). Isolated larvae from treated pigs were orally inoculated into mice to assess viability of ML from each treatment. Only Mebendazole treatment of pigs significantly reduced ML viability in mice. The effect of timing of the effective Mebendazole treatment on ML from a longer term infection was then examined in a second experiment. Analysis revealed that Mebendazole treatment of pigs with 250mg/kg over 3 days (83mg/kg/day) or 5 days (50mg/kg/day) reduced numbers of ML recovered from pig tissues compared to untreated, infected controls, and rendered ML non-infective to mice; Mebendazole treatment of pigs with 250mg/kg in a single dose was not effective in reducing ML numbers recovered from pigs or in impacting ML infectivity to mice. An examination of the lowest effective dose of Mebendazole on encysted ML was determined in a third experiment. Mebendazole of pigs with 5, 50, or 100mg/kg over 3 days demonstrated that 5 or 50mg/kg over 3 days insufficient to reduce infectivity in recovered ML, while 100mg/kg (and 83g from experiment 2) over 3 days significantly reduces infectivity of ML. This procedure provides a means to evaluate the efficacy of various anthelmintic treatments on the viability of Trichinella spiralis ML in pig tissues, and identified Mebendazole, at 83-100mg/kg administered over a 3-5 day period as an anthelmintic which renders encysted Trichinella spiralis ML from pig tissues non-infective. As risk from Trichinella significantly impacts acceptance of pork from pasture-raised pigs, these data provide a method, especially for producers of these high-risk pigs, to eliminate the potential of Trichinella transmission from infected pork.

Composite scores, where the results of two or more measures are combined, are commonly used in many fields, including ethology. Composite scores can simplify the analysis and interpretation of data while capturing the salient features of the underlying latent variable(s) approximated by the score. Here we outline four approaches for constructing composite scores in ethological studies: ad hoc (AH) assignment, discriminant analysis (DA), principal components analysis (PCA), and partial least squares (PLS). We give examples of each using previously published data from a study of responses of lone star ticks ( Amblyomma americanum ) to several deterrent phytochemicals. In most cases, researchers construct AH composite scores by subjectively assigning weights and signs to the behavioral components; unity weighting constrains weights to −1 or 1 on standardized variables. Because the weights and signs of the coefficients are subjectively assigned, AH scores may generate a spurious result. DA can be used to construct composite behavioral scores when there are clearly defined treatments or preference tests using distinct stimuli. The DA score created consists of orthogonal variables that capture the variability in the behavioral measures most closely aligned with the differences among treatment or stimuli variables. This approach assumes that subjects discriminate treatment or stimuli differences, but may not manifest clear overt behavior that they are able to do so; it reduces dimensionality, usually to a single axis, representing the underlying latent variable of interest. The PCA approach is similar to DA except that the composite score is created independently of treatment or stimuli variables. Thus, this method can be used to investigate possible relationships between a composite score and any relevant independent variable, perhaps measured asynchronously with the behaviors. PLS is a multivariate method related to DA and PCA and is also used to create latent orthogonal variables. However, these new variables are constructed to maximize correlation with one or more continuous independent variables. Creation of a composite score requires the researcher to consider not only the method used to create it, but, at an earlier stage in the research, which behaviors should be components and how best to measure them.

Experimental statistics are a key element for innovation in the agricultural sector. Commonly used statistical methods in experimentation are relatively simple, reliable, and widely used. However, the many problems in the quality of statistical analyses reported in the agricultural science literature highlight a need for continuing discussion on and updating of this topic. This article reviews critical points about classic linear models procedures commonly used in agricultural statistics, frequent procedures in publications in the agricultural sciences. Due to the evolution of statistical science some common recommendations from the past should no longer be followed.

Foodborne pathogens continue to pose a public health risk and can cause serious illness and outbreaks of disease in consumers. The consumption of raw or undercooked infected meat, such as pork containing infectious stages of Toxoplasma gondii, may be a major route of transmission to humans. Given the occasional presence of T. gondii in pork meat and the frequent use of pork for products not intended to be cooked, such as dry-cured ham, a potential risk exists for T. gondii transmission to consumers of these products. The purpose of this study was to determine the seroprevalence of T. gondii in U.S. market hogs and sows at slaughter. A total of 20,209 sera samples collected from 22 U.S. slaughterhouses, including 15 of the top 25 largest slaughter plants in the United States, were tested for T. gondii antibodies using a commercial ELISA assay. Seroprevalence in this study was 0.74%, with a herd prevalence of 10.86%. We compared seroprevalence of T. gondii in market hogs vs. sows from a separate but geographically similar set of slaughterhouse locations, with serum samples screened using the T. gondii modified agglutination test. This set of market hogs demonstrated 0% seroprevalence for T. gondii, while sows from geographically similar but separate slaughter facilities demonstrated a seroprevalence of 1.03%. Overall, both analyses show low seroprevalence of T. gondii in U.S market hogs and sows, respectively, and a marked drop in prevalence in market hogs and sows compared to previous studies.

Brassica vegetables may modulate cancer risk by regulation of xenobiotic metabolizing enzymes (XMEs). In a randomized crossover study, the effect of kale consumption on CYP1A2, CYP2A6, XO, and NAT2 activity was determined by urinary caffeine metabolite ratios, UGT1A1 activity by serum bilirubin concentrations, and GSTA protein and GST activity in blood by ELISA. Adults (n = 25) consumed a basal diet supplemented with kale and radish for 14 days or control vegetables. The kale diet increased CYP1A2 activity by 16.4% on day 8 and 15.2% on day 15 compared to control. Conjugated bilirubin was reduced by the kale diet, decreasing from 19.4 to 14.3 to 9.5% of total bilirubin on days 1, 8, and 15, respectively, which may be explained by induction of MRP2. Other XMEs were not affected by diet. The implications of these results for cancer risk will be clarified as the functions of these XMEs become better understood.

Populations of host-seeking blacklegged tick, Ixodes scapularis (Say) and lone star tick, Amblyomma americanum (L.), nymphs were monitored at selected sites for 4 yr and at additional randomly selected sites in the Greenbelt National Park, MD for two of those years. Ticks collected from the random sites during the second year of the study were tested for the presence of human pathogens. Borrelia burgdorferi Johnson et al. was detected in 22.2% of the I. scapularis nymphs collected while Anaplasma phagocytophilum Foggie (Dumler et al.) was detected in 3.7%, and one nymph was coinfected with both pathogens. No I. scapularis nymphs tested positive for Babesia microti (Franca) and no A. americanum nymphs tested positive for Ehrlichia spp. In the years when both random and nonrandom sites were sampled (sampled <= 2 d apart, n = 14 d), significantly more A. americanum nymphs (P = 0.003) were captured at the nonrandom sites than at the random sites; no difference (P = 0.2415) was found for I. scapularis nymphs. No density effect due to vegetational communities was found for nymphs of either species of tick. Host-seeking nymphs of both species of ticks were abundant the first year of flag sampling, dropped dramatically in numbers the second year, and gradually increased ( particularly A. americanum) the following 2 yr. The annual variations in tick densities demonstrate the value of early season-monitoring of tick populations on park premises, which affords park managers an opportunity to take appropriate measures in the event of a year of high tick abundance.

A key characteristic of scientific research is that the entire experiment (or series of experiments), including the data analyses, is reproducible. This aspect of science is increasingly emphasized. The Materials and Methods section of a scientific paper typically contains the necessary information for the research to be replicated and expanded on by other scientists. Important components are descriptions of the study design, data collection, and statistical analysis of those data, including the software used. In the Results section, statistical analyses are presented; these are usually best absorbed from figures. Model parameter estimates (including variances) and effect sizes should also be included in this section, not just results of significance tests, because they are needed for subsequent power and meta-analyses. In this article, we give key components to include in the descriptions of study design and analysis, and discuss data interpretation and presentation with examples from the horticultural sciences.

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BACKGROUNDThe National Health and Nutrition Examination Survey physical activity questionnaire (PAQ) is used to estimate activity energy expenditure (AEE) and moderate to vigorous physical activity (MVPA). Bias and variance in estimates of AEE and MVPA from the PAQ have not been described, nor the impact of measurement error when utilizing the PAQ to predict biomarkers and categorize individuals.METHODSThe PAQ was administered to 385 adults to estimate AEE (AEE:PAQ) and MVPA (MVPA:PAQ), while simultaneously measuring AEE with doubly labeled water (DLW; AEE:DLW) and MVPA with an accelerometer (MVPA:A).RESULTSAlthough AEE:PAQ [3.4 (2.2) MJ·d-1] was not significantly different from AEE:DLW [3.6 (1.6) MJ·d-1; P > .14], MVPA:PAQ [36.2 (24.4) min·d-1] was significantly higher than MVPA:A [8.0 (10.4) min·d-1; P < .0001]. AEE:PAQ regressed on AEE:DLW and MVPA:PAQ regressed on MVPA:A yielded not only significant positive relationships but also large residual variances. The relationships between AEE and MVPA, and 10 of the 12 biomarkers were underestimated by the PAQ. When compared with accelerometers, the PAQ overestimated the number of participants who met the Physical Activity Guidelines for Americans.CONCLUSIONSGroup-level bias in AEE:PAQ was small, but large for MVPA:PAQ. Poor within-participant estimates of AEE:PAQ and MVPA:PAQ lead to attenuated relationships with biomarkers and misclassifications of participants who met or who did not meet the Physical Activity Guidelines for Americans.

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent of DBF. The DBF cream was tested against nymphs of lone star tick, Amblyomma americanum (L.). In vertical filter paper assays, the three extracts applied at 0.413 mg extract/cm2 filter paper repelled 96.7–100 % of brown dog tick, Rhipicephalus sanguineus (Latreille) nymphs, whereas, at the same rate, only one extract repelled >90 % A. americanum nymphs. Adult (mixed sexes) American dog ticks, Dermacentor variabilis (Say), required a higher concentration to be repelled effectively; two extracts, applied at 0.827 mg extract/cm2 filter paper, repelled ≥90 % of the D. variabilis. In contrast, all extracts applied at much lower concentration (0.206 mg extract/cm2 filter paper) repelled 100 % adult blacklegged ticks, Ixodes scapularis Say (only females tested). Of the two more repellent extracts, one lost most of its activity against A. americanum nymphs in <4 h when applied at 0.827 mg extract/cm2 filter paper, whereas the other repelled 66.7 % of the nymphs at 192 h after application. At 0.206 mg extract/cm2 filter paper, one extract was as repellent as deet against A. americanum nymphs. In a vertical bioassay in which nylon organdy was substituted for filter paper, DBF, at the rates of 1.67 and 0.835 mg cream/cm2, repelled 76.7 and 30.0 % A. americanum nymphs, respectively. These findings indicate that when applied appropriately DBF should afford some protection to canines against tick bites.

We examined all articles in volume 139 and the first issue of volume 140 of the Journal of the American Society for Horticultural Science (JASHS) for statistical problems. Slightly fewer than half appeared to have problems. This is consistent with what has been found for other biological journals. Problems ranged from inappropriate analyses and statistical procedures to insufficient (or complete lack of) information on how the analyses were performed. A common problem arose from taking many measurements from the same plant, which leads to correlated test results, ignored when declaring significance at P = 0.05 for each test. In this case, experiment-wise error control is lacking. We believe that many of these problems could and should have been caught in the writing or review process; i.e., identifying them did not require an extensive statistics background. This suggests that authors and reviewers have not absorbed nor kept current with many of the statistical basics needed for understanding their own data, for conducting proper statistical analyses, and for communicating their results. For a variety of reasons, graduate training in statistics for horticulture majors appears inadequate; we suggest that researchers in this field actively seek out opportunities to improve and update their statistical knowledge throughout their careers and engage a statistician as a collaborator early when unfamiliar methods are needed to design or analyze a research study. In addition, the ASHS, which publishes three journals, should assist authors, reviewers, and editors by recognizing and supporting the need for continuing education in quantitative literacy.

Boxwood blight caused by Calonectria pseudonaviculata is a newly emergent disease of boxwood (Buxus spp. L.) in the United States that causes leaf drop, stem lesions, and plant death. A rapid and reliable laboratory assay that enables screening hundreds of boxwood genotypes for resistance to boxwood blight is needed to enable breeding and selection of resistant cultivars. Using eight boxwood cultivars with differing susceptibilities, we examined parameters for a screening assay comparing whole plant inoculation with detached leaf inoculation, use of mycelium versus spores as the inoculum, comparison of times of the year for inoculation, and comparison of two leaf inoculation methods. Inoculation of detached leaves gave comparable results to inoculation of whole plants when compared across genotypes, although the detached leaf assay resulted in greater percentages of symptom expression. The time of year of plant inoculation (spring, summer, or winter) did not affect the relative expression of symptoms among the most resistant and susceptible genotypes. Inoculating plants with mycelium was as effective as spore inoculation for causing disease symptoms and allowed us to distinguish the more resistant genotypes, yet mycelium inoculation was much easier to prepare in large quantities for multiple assays.

Sound design of experiments combined with proper implementation of appropriate statistical methods for data analysis are critical for producing meaningful scientific results that are both replicable and reproducible. This communication addresses specific aspects of design and analysis of experiments relevant to the dairy sciences and, in so doing, responds to recent concerns raised in a letter to the editor of the Journal of Dairy Science regarding journal policy for research publications on pen-based animal studies. We further elaborate on points raised, rectify interpretation of important concepts, and show how aspects of statistical inference and elicitation of research conclusions are affected.

BACKGROUND:The number of days of data and number of subjects necessary to estimate total physical activity (TPA) and moderate-to-vigorous physical activity (MVPA) requires an understanding of within- and between-subject variances, and the influence of sex, body composition, and age.METHODS:Seventy-one adults wore accelerometers for 7-day intervals over 6 consecutive months.RESULTS:Body fat and sex influenced TPA and MVPA. The sources of subject-related variation for TPA and MVPA were within-subject (48.4% and 54.3%), between-subject (34.3% and 31.8%), and calendar effects (17.3% and 13.9%). Based on within-subject variances, the error associated with estimating TPA and MVPA by collecting 1 to 7 days of data ranged from 28.2% to 13.3% for TPA and 62.0% to 28.6% for MVPA. Based on between-subject variances, detecting a 10% difference between 2 groups at a power of 90% requires approximately 200 and 725 subjects per group for TPA and MVPA, respectively.CONCLUSIONS:Estimates of MVPA are more variable than TPA in overweight adults, therefore more days of data are required to estimate MVPA and larger sample sizes to detect treatment differences for MVPA. Log-transforming data reduces the need for additional days of data collection, thereby improving chances of detecting treatment effects.

Reducing the number of animal subjects used in biomedical experiments is desirable for ethical and practical reasons. Previous reviews of the benefits of reducing sample sizes have focused on improving experimental designs and methods of statistical analysis, but reducing the size of control groups has been considered rarely. We discuss how the number of current control animals can be reduced, without loss of statistical power, by incorporating information from historical controls, i.e. subjects used as controls in similar previous experiments. Using example data from published reports, we describe how to incorporate information from historical controls under a range of assumptions that might be made in biomedical experiments. Assuming more similarities between historical and current controls yields higher savings and allows the use of smaller current control groups. We conducted simulations, based on typical designs and sample sizes, to quantify how different assumptions about historical controls affect the power of statistical tests. We show that, under our simulation conditions, the number of current control subjects can be reduced by more than half by including historical controls in the analyses. In other experimental scenarios, control groups may be unnecessary. Paying attention to both the function and to the statistical requirements of control groups would result in reducing the total number of animals used in experiments, saving time, effort and money, and bringing research with animals within ethically acceptable bounds.

Behavioral bioassays remain a standard tool in the discovery, development, and registration of arthropod repellents. Tick repellent bioassays are generally uncomplicated, but their results can be affected by basic variables (e.g., dimensions of testing materials, substrate, timing, temperature) of the assay. Using lone star tick, Amblyomma americanum (L.), nymphs in climbing bioassays, we tested for the effects of substrate, solvent, and drying time on tick responses. In dose-response tests, the widely used repellents N,N-diethyl-3-methyl benzamide (deet) and 1-methyl-propyl-2-(hydroxyethyl)-1-piperidinecarboxylate (picaridin) were applied to filter paper strips and challenged by ticks at 10, 20, 30, 40, and 120 min after application. At 10-min drying time, repellency at the intermediate concentration 500 nmol repellent/cm2 filter paper was significantly lower for ethanol solutions of deet and picaridin (0 and 10% ticks repelled, respectively) than for solutions of deet and picaridin in acetone (96.7 and 76.7% ticks repelled, respectively). Repellency was greatest for both the acetone and ethanol solutions of deet and picaridin when challenged 120 min after application, and at shorter drying times at the highest concentration tested (2,000 nmol compound/ cm2). The repellency of picaridin relative to deet differed at some combinations of solvent and drying time but not others. In dose-response tests using different paper substrates and a drying time of 10 min, both ethanol and acetone solutions of deet differed in repellency, depending on both the paper substrate and the solvent. However, there were no differences in repellency between ethanol and acetone solutions of deet applied to nylon organdy in an in vitro and in an in vivo (fingertip) bioassay. When deet in solution with various proportions of ethanol:water was applied at 2,000 nmol deet/cm2 filter paper, the proportion of ticks repelled decreased as the proportion of water in the test solutions increased. Somewhat similar results were seen for solutions of deet in an acetone solvent. Water absorbed from the atmosphere may affect the efficacy of repellents in solution with anhydrous ethanol. Overall, results obtained from bioassays that differ in seemingly minor ways can be surprisingly different, diminishing the value of comparing studies that used similar, but not identical, methods. Nylon organdy or another similar thin cloth may be preferable to filter papers and copier paper for minimizing solvent-related differences. When a paper substrate is used, acetone may be the more suitable solvent if the solubility of the test compound and other factors allow.

SummaryIn this article we provide guidelines on statistical design and analysis of data for all kinds of honey bee research. Guidelines and selection of different methods presented are, at least partly, based on experience. This article can be used: to identify the most suitable analysis for the type of data collected; to optimise one's experimental design based on the experimental factors to be investigated, samples to be analysed, and the type of data produced; to determine how, where, and when to sample bees from colonies; or just to inspire. Also included are guidelines on presentation and reporting of data, as well as where to find help and which types of software could be useful.