Reference Value Advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel

Abstract: International recommendations for determination of reference intervals have been recently updated, especially for small reference sample groups, and use of the robust method and Box–Cox transformation is now recommended. Unfortunately, these methods are not included in most software programs used for data analysis by clinical laboratories. We have created a set of macroinstructions, named Reference Value Advisor, for use in Microsoft Excel to calculate reference limits applying different methods. For any series of data, Reference Value Advisor calculates reference limits (with 90% confidence intervals [CI]) using a nonparametric method when n≥40 and by parametric and robust methods from native and Box–Cox transformed values; tests normality of distributions using the Anderson–Darling test and outliers using Tukey and Dixon–Reed tests; displays the distribution of values in dot plots and histograms and constructs Q–Q plots for visual inspection of normality; and provides minimal guidelines in the form of comments based on international recommendations. The critical steps in determination of reference intervals are correct selection of as many reference individuals as possible and analysis of specimens in controlled preanalytical and analytical conditions. Computing tools cannot compensate for flaws in selection and size of the reference sample group and handling and analysis of samples. However, if those steps are performed properly, Reference Value Advisor, available as freeware at http://www.biostat.envt.fr/spip/spip.php?article63 , permits rapid assessment and comparison of results calculated using different methods, including currently unavailable methods. This allows for selection of the most appropriate method, especially as the program provides the CI of limits. It should be useful in veterinary clinical pathology when only small reference sample groups are available.     

Canine reference intervals for the Sysmex XT-2000iV hematology analyzer

Background: The laser‐based Sysmex XT‐2000iV hematology analyzer is increasingly used in veterinary clinical pathology laboratories, and instrument‐specific reference intervals for dogs are not available. Objective: The purpose of this study was to establish canine hematologic reference intervals according to International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines using the Sysmex XT‐2000iV hematology analyzer. Methods: Blood samples from 132 healthy purebred dogs from France, selected to represent the most prevalent canine breeds in France, were analyzed. Blood smears were scored for platelet (PLT) aggregates. Reference intervals were established using the nonparametric method. PLT and RBC counts obtained by impedance and optical methods were compared. Effects of sex and age on reference intervals were determined. Results: The correlation between impedance (I) and optical (O) measurements of RBC and PLT counts was excellent (Pearson r=.99 and .98, respectively); however, there were significant differences between the 2 methods (Student's paired t‐test, P<.0001). Differences between sexes were not significant except for HCT, PLT‐I, and PLT‐O. WBC, lymphocyte, and neutrophil counts decreased significantly with age (ANOVA, P<.05). Median eosinophil counts were higher in Brittany Spaniels (1.87 × 10⁹/L), Rottweilers (1.41 × 10⁹/L), and German Shepherd dogs (1.38 × 10⁹/L) than in the overall population (0.9 × 10⁹/L). PLT aggregates were responsible for lower PLT counts by the impedance, but not the optical, method. Conclusion: Reference intervals for hematologic analytes and indices were determined under controlled preanalytical and analytical conditions for a well‐characterized population of dogs according to international recommendations.     

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).     

Mechanical comparison of two suture constructs for extra-capsular stifle stabilization

Objective: Mechanical evaluation of 2 suture constructs for extracapsular stifle stabilization. Study Design: In vitro study. Sample Population: Crimped interlocking loop constructs (ILC) of 45 kg nylon leader line (NLL) and Orthofiber^® (OF). Methods: ILC were tightened to 100 N, then crimp secured. Ramp to failure (n=10/group)—Data were derived from force/displacement plots. Stress–relaxation testing (n=10/group)—ILC's were nondestructively loaded and held at resultant displacement as force data were recorded. Incremental, cyclic loading (n=10/group)—ILC's were loaded (5 cycles/set) starting at 100 N and incrementally increased by 50 N (1 and 3 Hz protocols). Loop tension and elongation were recorded after each set. Results: Ramp to failure—initial loop tension was similar (NLL 75.5 ± 9.5 N; OF 68.7 ± 10.4 N, P=.140). Tested OF constructs were stiffer (NLL 125.7 ± 4.0; OF 234.6 ± 25.0 N/mm, P≤.001), had lower yield load (NLL 193.6 ± 13.8; OF 137.3 ± 94.3 N, P≤.001), lower peak load (NLL 873.7 ± 68.6; OF 653.6 ± 80.2 N, P≤.001), and lower elongation at failure (NLL 19.1 ± 1.4; OF 5.2 ± 1.0 mm, P≤.001) and at yield (NLL 1.52 ± 0.2; OF 0.3 ± 0.6 mm, P=.003) than NLL constructs. Yield in NLL ILC's was variable knot tightening/crimp slippage, but only crimp‐suture slippage in OF. Stress–relaxation testing—OF demonstrated greater relaxation. Incremental, cyclic loading—induced ILC elongation and tension loss in both groups, independent of loading frequency. NLL lost tension at lower rate, but elongated more than OF. Conclusions: NLL construct is mechanically superior to OF construct.     

Leishmania chagasi infection in cats with dermatologic lesions from an endemic area of visceral leishmaniosis in Brazil

Although dogs are considered the main domestic reservoirs for Visceral Leishmaniosis (VL), which is caused in the Americas by Leishmania chagasi, infected cats have also been recently found in endemic areas of several countries and became a public health concern. Accordingly, the purpose of this study was to evaluate cats with dermatologic lesions from an endemic area of VL and the natural infection of L. chagasi. A total of 55 cats were selected between April 2008 and November 2009 from two major animal shelters of Ara?atuba, Southeastern Brazil. All cats underwent general and dermatologic examinations, followed by direct parasitological examination of lymphoid organs, immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT). In addition, detection of amastigotes was performed by immunohistochemistry (IHC) in skin lesions of all cats. VL was diagnosed in 27/55 (49.1%) cats with dermatological problems. Amastigotes were found in lymphoid organs of 10/27 (37.0%) cats; serology of 14/27 (51.9%), 6/27 (22.2%) and 5/27 (18.5%) cats was positive for ELISA, IFAT and both, respectively. The IHC identified 9/27 (33.3%) cats; 5/27 (18.5%) were positive only for IHC and therefore increased the overall sensitivity. Specific FIV antibodies were found in 6/55 (10.9%) cats, of which 5/6 (83.3%) had leishmaniosis. Real time PCR followed by amplicon sequencing successfully confirmed L. chagasi infection. In conclusion, dermatological lesions in cats from endemic areas was highly associated to visceral leishmaniosis, and therefore skin IHC and differential diagnosis of LV should be always conducted in dermatological patients in such areas.     

BTV infection in wild ruminants, with emphasis on red deer: a review

The distribution of bluetongue virus has changed, possibly related to climate change. Vaccination of domestic ruminants is taking place throughout Europe to control BT expansion. The high density of wild red deer (Cervus elaphus) in some European regions has raised concerns about the potential role that unvaccinated European wild ungulates might play in maintaining or spreading the virus. Most species of wild ruminants are susceptible to BTV infection, although frequently asymptomatically. The red deer population density in Europe is similar to that of domestic livestock in some areas, and red deer could account for a significant percentage of the BTV-infection susceptible ruminant population in certain regions. High serum antibody prevalence has been found in red deer, and BTV RNA (BTV-1, BTV-4 and BTV-8) has been repeatedly detected in naturally infected European red deer by means of RT-PCR. Moreover, red deer may carry the virus asymptomatically for long periods. Epidemiological studies suggest that there are more BT cases in domestic ungulates in those areas where red deer are present. Vector and host density and environmental factors are implicated in the spatial distribution of BT. As in domestic ruminants, BTV transmission among wild ruminants depends almost exclusively on Culicoides vectors, mainly C. imicola but also members of the C. obsoletus and C. pulicaris complex. However, BTV transmission from red deer to the vector remains to be demonstrated. Transplacental, oral, and mechanical transmissions are also suspected. Thus, wild red deer contribute to the still unclear epidemiology of BTV in Europe, and could complicate BTV control in domestic ruminants. However, further research at the wildlife host-vector-pathogen interface and regarding the epidemiology of BT and BT vectors in wildlife habitats is needed to confirm this hypothesis. Moreover, red deer could be used as BT sentinels. Serum and spleen tissue of calves sampled from late autumn onwards should be the target samples when establishing a BTV surveillance program.     

Spatial evaluation of soil salinity using the WET sensor in the irrigated area of the Segura river lowland

The electrical conductivity of the water within the soil pores (EC_p) measured with the WET sensor, appears to be a reliable estimate of soil salinity. A methodology combining the use of the WET sensor along with geostatistics was developed to delimit and evaluate soil salinity within an irrigated area under arid to semiarid Mediterranean climate in SE Spain. A systematic random sampling of 104 points was carried out. The association between EC_p and the saturation‐extract electrical conductivity (EC_se) was assessed by means of correlation analysis. The semivariograms for EC_p were obtained at three different soil depths. Interpolation techniques, such as ordinary kriging and cokriging, were applied to obtain EC_p levels in the unknown places. For each one of the soil depths, a model able to predict EC_se from EC_p was developed by means of ordinary least squares regression analysis. A good correlation (r = 0.818, p < 0.001) between EC_p and EC_se was found. Spherical spatial distribution was the best model to fit to experimental semivariograms of EC_p at 10, 30, and 50 cm soil depths. Nevertheless, cokriging using the EC_p of an adjacent soil depth as an auxiliary variable provided the best results, compared to ordinary kriging. An analytical propagation‐error methodology was found to be useful to ascertain the contribution of the spatial interpolation and ordinary least squares analysis to the uncertainty of the EC_se mapping. This methodology allowed us to identify 98% of the study area as affected by salinity problems within a rooting depth of 50 cm, with the threshold of EC_se value at 2 dS m^–1. However, considering the crops actually grown and 10% potential reduction yield, the soil‐salinity‐affected area decreased to 83%. The use of sensors to measure soil salinity in combination with geostatistics is a cost‐effective way to draw maps of soil salinity at regional scale. This methodology is applicable to other agricultural irrigated areas under risk of salinization.     

Nutrient status of apple leaves not affected by three years of irrigation using partial rootzone drying

Partial rootzone drying (PRD) is a water‐saving irrigation technology that may affect apple (Malus domestica Borkh cv. Golden Delicious/Malling7)‐tree nutrition if applied for an extended period. The objective of this study was to test the hypothesis that long‐term application of PRD causes seasonal changes in macro‐ and micronutrients of apple leaves. The irrigation treatments were: (1) commercial irrigation as control (CI) and (2) PRD. After 3 years of evaluation, PRD irrigation had saved about 3240 m³ of water per hectare. Leaf xylem water potential was slightly lower in the PRD treatment than in CI. The seasonal concentration of macro‐ and micronutrients was comparable between treatments, although significant differences were found at times. The macronutrient concentrations were within the normal range in PRD apple leaves. All micronutrient concentrations were slightly above the normal range except for Zn, which was slightly below the normal range. No physiological disorders associated with plant nutrition were observed on leaves or fruits. Therefore, data suggest that PRD did not alter apple‐tree nutrition during the 3‐year trial. Thus, PRD may be feasible for apple production in Central Mexico. However, further studies need to be conducted in those regions where groundwater is the main water source for irrigation and rain is negligible, particularly during the growing season.     

The under-recognized dominance of Verrucomicrobia in soil bacterial communities

Verrucomicrobia are ubiquitous in soil, but members of this bacterial phylum are thought to be present at low frequency in soil, with few studies focusing specifically on verrucomicrobial abundance, diversity, and distribution. Here we used barcoded pyrosequencing to analyze verrucomicrobial communities in surface soils collected across a range of biomes in Antarctica, Europe, and the Americas (112 samples), as well as soils collected from pits dug in a montane coniferous forest (69 samples). Data collected from surface horizons indicate that Verrucomicrobia average 23% of bacterial sequences, making them far more abundant than had been estimated. We show that this underestimation is likely due to primer bias, as many of the commonly used PCR primers appear to exclude verrucomicrobial 16S rRNA genes during amplification. Verrucomicrobia were detected in 180 out of 181 soils examined, with members of the class Spartobacteria dominating verrucomicrobial communities in nearly all biomes and soil depths. The relative abundance of Verrucomicrobia was highest in grasslands and in subsurface soil horizons, where they were often the dominant bacterial phylum. Although their ecology remains poorly understood, Verrucomicrobia appear to be dominant in many soil bacterial communities across the globe, making additional research on their ecology clearly necessary.