Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

45阶Kirkman三连系的构造与计数

给出了边矩阵和完全三分图的定义,提出了利用边矩阵、完全三分图构造45阶Kirkman三连系的思路。介绍了不同构的45阶Kirkman三连系构造的全过程,讨论了45阶Kirkman三连系的计数。     

Microbial Composition before and after Conservation of Grass-Dominated Haylage Harvested Early,Middle, and Late in the Season

Haylage for horses is often harvested in late plant maturity, which could be associated with an increased risk of impaired hygienic quality in the forage and short aerobic storage stability after bale opening, but knowledge in this area is scant. An experiment was conducted in which the microbial composition was analyzed before and after conservation of primary growth haylage harvested early (June), middle (July), and late (August) in the season during 1 year. The counts of yeast, enterobacteria, and lactic acid bacteria (LAB) in preconserved herbage increased with the advancing harvest time (P ≤ .02). After conservation, the August haylage had increased counts of enterobacteria (log 4.3 colony-forming unit [CFU]/g) and LAB (log 6.5 CFU/g), compared with the June and July haylage (log ≤1.7 CFU/g for enterobacteria and ≤5.7 CFU/g for LAB, P < .001). The yeast counts were the lowest in the June haylage (log 5.0 CFU/g) compared with the July and August haylage (log ≥6.3 CFU/g, P < .001). After conservation, the mold counts were lower in the June haylage and greater in the August haylage (P = .01). In the preconserved herbage, Cladosporium cladosporioides was the most common mold species in June but Fusarium poae was in July, and Mucor fragilis in August. After conservation, Penicillium carneum was the only species found in the June haylage, with M. circinelloides most frequently found in the July haylage and M. hiemalis and M. circinelloides found at similar frequencies in the August haylage. An advanced harvest time resulted in greater counts of enterobacteria, yeast, and LAB and an increased number of mold species in the conserved haylage. The aerobic storage stability of the opened haylage bales measured by temperature was similar among the harvests.     

Effect of a short duration feed withdrawal followed by full feeding on marbling fat in beef carcasses

The effect of feed withdrawal for 48 h, prior to initiation of the finishing (fattening) period (75 d) on carcass marbling fat was studied in 120 European × British cross-bred heifers with an average weight of 585 ± 39 kg. Heifers were randomized in a 2 × 2 factorial design experiment with two dietary management treatments, where half the heifers were provided the feed components of steam rolled barley and barley silage either free choice or as a total mixed ration (TMR) containing 87% steam rolled barley and 13% barley silage with ad libitum vitamins and minerals via salt blocks for all animals. Within each dietary management treatment, 30 heifers were denied feed (water was available) for 48 h prior to the two week adaptation to the high grain diet preceding the 75 d finishing period. At the end of the 48 h feed denial blood samples were collected from the jugular vein prior to feeding for determination of glucose and insulin concentrations, which indicated that 48 h feed withdrawal consistently decreased (P = 0.0001) plasma concentrations of both glucose and insulin but the ratios of the concentrations of glucose to insulin were not affected. At slaughter samples of subcutaneous fat from the brisket (BF) and skirt muscle (pars costalis diaphragmatis; PCD) were procured for determination of chemical fat content, fat dissected from the muscle and for enumeration of adipocytes, less than 35 μm in diameter and to determine the average cell size in the dissected fat and from the BF by flow-cytometry of adipocytes fixed in osmium tetroxide. The carcass characteristics were also obtained. Although no differences due feed withdrawal for 48 h were evident for carcass weight, percent lean (saleable) meat yield, rib eye area, average fat cover, fat content of PCD or BF, the US marbling score was increased (P = 0.048) and the amount of dissected fat from the muscle tended to be higher (P = 0.107), thus 81% of the carcasses graded “US Choice” or “Canada AAA,” or displayed at least a “small” amount of intramuscular fat as compared (P = 0.0807) to 68% of the heifers not denied feed. Based on more than three years of weekly prices of carcasses that graded “Canada AAA” and “Canada AA,” these experimental results suggested that the expected price of a finished heifer could increase by $4.61 Canadian if a 48 h feed withdrawal was imposed prior to initiation of the finishing phase. Although significant differences in adipocyte numbers due to a single time 48 h feed withdrawal prior to initiation of the finishing phase were not detected, the carcass quality factors were affected leading to an odds ratio of 1.84 times in favour of cattle carcasses to grade “Canada AAA” than if fed continuously.     

计数资料参数差异的显著性检验

根据多项分布，在大样本条件下，通过构造Ｔ统计量，实现了计数资料参数差异的显著性检验，现行的百分率ｔ（ｕ）检验只是该检验方法的一种特殊形式。     

Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria

ABSTRACT:   The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.     

Methods of analysis for georeferenced sample counts of tarnished plant bugs in cotton

The problem of analyzing georeferenced cotton pest insect samples when a large percentage of the counts are zero is examined. The use of appropriate statistical methods for their analysis is required. To demonstrate this, georeferenced samples (n = 63) of tarnished plant bugs (TPBs; Lygus lineolaris [Palisot de Beauvois] (Heteroptera: Miridae)) were analyzed by three statistical methods and the results were compared. Correlation analysis of the sample counts with 25 classes of cotton growth derived from an unsupervized classification of multispectral imagery was followed by a complete enumeration analysis comprising three scenarios. The first scenario assumed the insect samples were unstratified. A distribution of sample averages was created by complete enumeration of all combinations of samples taken four at a time. The second scenario used imagery of the cotton fields to allocate the samples among three cotton growth categories (marginal, good or best) derived by a supervized classification of the 25 unsupervized classes. The insect samples associated with these categorical habitats were completely enumerated using allocations of 4, 6, 8 or 10 samples at a time from various sample sizes to determine how different allocations affected the results. The mean was not affected, but the standard deviation decreased with increased allocation sizes in all habitats. The third scenario used the two observers and three habitat categories to create six additional complete enumeration distributions by allocating four samples at a time from groups of varying sample sizes. These enumeration distributions are non-parametric estimators of the sampling distribution of: (1) the sample averages of a given sample size when samples are taken from the entire field, (2) the sample averages of a given size when samples are taken from each cotton habitat or (3) the sample averages of a given sample size from samples taken from each habitat by each observer. To support the enumeration analyses, these insect samples were analyzed further by Poisson regression models. These models showed significant differences between TPB counts by the two observers and among the habitats, whereas the observer by habitat interaction was not significant. For every combination of observer and cotton growth category, a Poisson regression model estimated the mean rate of TPB numbers. These means were similar to the corresponding modes of the complete enumeration distributions. The two non-standard methods showed that TPB numbers differed by habitat categories even though there were samples with a zero count, whereas a correlation analysis failed to identify a relationship between TPB sample counts and unsupervized habitat classes.     

枚举法与动态规划法结合优化田间管网

根据新疆农八师垦区滴灌大田作物机械化作业的特点，在一个灌区内选用相同管径的毛管，以一条干管所辖面积为一单元，建立了田间管网的优化模型，并给出了解法及优化算例。     

土壤真菌分离和计数方法的探讨

综述了分离土壤真菌的方法以及测定土壤真菌的间接计数法和直接计数法;介绍了常见的分离和计数培养基;评价了各种方法和培养基的优缺点;并着重讨论了各种分离和计数方法的应用。     

The genus Amygdalus L. (Rosaceae): Species relationships, distribution and evolution under domestication

Summary The paper reviews the taxonomic relationships, morphological distinction, geographical distribution and ecological specificities of the twenty six species recognized (by us) in Amygdalus L. It also surveys the intra-genetic structure in this genus stressing the fact that they fall into five groups of closely related vicarious species: (i) Communis group (9 species), (ii) Orientalis group (6 species), (iii) Sect. Chamaeamygdalus (4 species), (iv) Sect. Spartioides (2 species), and (v) Subgenus Dodecandara (5 species). Within each group, species are separated from one another geographically (in few cases by altitude). The only major exception to such allopatric distribution is found in A. communis L., and this is interpreted as an outcome of domestication. Interspecific sterility barriers are absent, or only weakly developed in Amygdalus, and numerous inter-specific hybrids (particularly between the crop and various wild almond species) have been detected. Therefore most (may be all) wild almond species constitute the primary gene-pool of the cultivated nut crop.The combined botanical and archaeological evidene points to the Levant countries as the place where the almond was taken into cultivation. Wild forms of A. communis are very likely native only to this area. Also the earliest archaeological signs of almond domestication come from this area.