Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Humic acids are ubiquitous and abundant in terrestrial environments; therefore, they are often co-extracted with nucleic acids and interfere with quantitative PCR (qPCR) assays. In this study a recently developed NanoGene assay that is resistant to interference by humic acids was evaluated for gene detection in soil samples. The NanoGene assay utilizes a combination of magnetic beads, dual quantum dots labels, and DNA hybridization in solution. Seven soil samples containing different amounts of organic matter were tested to compare NanoGene and qPCR assays for their respective ability to detect a bacterial pathogen. We spiked the soils with Escherichia coli O157:H7, extracted genomic DNA, and conducted NanoGene and qPCR assays targeting the E. coli O157:H7-specific eaeA gene. To prevent the inhibition of PCR that is common when using DNA extracted from soils, we used a range of template DNA concentrations and BSA addition in the qPCR assay. Compared to the qPCR assay the NanoGene assay was significantly more resistant to the inhibitory effect of humic acids, successfully quantifying the eaeA gene within a linear (R² = 0.99) range of 10⁵ through 10⁸ CFU/g soil for all seven soil samples tested. In contrast, the qPCR assay was significantly inhibited using the same template DNA isolated from soils containing a range of organic content (2.0%–12%). Interestingly, the qPCR assay was still inhibited despite additional purification steps, suggesting that humic acids were still associated with DNA at a level that was inhibitory to qPCR. This study demonstrated that the NanoGene assay is suitable for quantitative gene detection in diverse soil types and is not susceptible to inhibition by humic acids and other organic compounds that commonly lead to false negative results in qPCR assays.     

Effects of the insecticide triazophos on the ultrastructure of the flight muscle of the brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae)

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a long-distance migratory insect pest in temperate eastern Asia and is a classical resurgent rice pest that is induced by insecticides. Knowledge of the effect of insecticides on the flight capacity of insects is needed to understand their migration, which may help to improve the management of insect pests. Our previous study demonstrated that some insecticides enhance the flight capacity of BPH. However, the effect of insecticides on the flight muscle of BPH is not well understood. Thus, the present study examined the effect of triazophos, a classical insecticide that induces the resurgence of BPH, on the ultrastructure of macropterous females using transmission electron microscopy. The results showed that the diameter of myofibrils one and two days after adult female emergence (DAE) following treatment with 40 ppm triazophos was significantly greater than that of the control, increasing by 31.4 and 21.5%, respectively. In addition, the length of the sarcomeres at two and three DAE was significantly greater than that of the control, increasing by 73.8 and 50.8%, respectively. The percentage of mitochondrial volume in the muscle fibres at one and two DAE was 146.2 and 67.7% greater than that of the control, respectively. These findings show that the mechanisms of triazophos-induced enhancement of the flight capacity of BPH involve changes in the ultrastructure of the flight muscle.     

Soybean yield and yield components as influenced by the single and repeated flaming

Field experiments were conducted to study the impact of single and multiple flaming on crop injury, yield components, and yield of soybean. The goal of this experiment was to determine the number of the maximum flaming treatments which soybean could tolerate without any yield loss. The treatments consisted of a non-flamed control, and broadcast flaming conducted one time (at VC-unfolded cotyledon, V2-second trifoliate, and V5-fifth trifoliate), two times (each at VC and V2, VC and V5, and V2 and V5 stages), and three times (at VC, V2, and V5 stages) resulting in a total of eight treatments. All plots were kept weed-free for the entire growing season by hand hoeing. A propane dose of 50 kg ha⁻¹ was applied with torches parallel to the crop row and at an operating speed of 4.8 km h⁻¹ for all treatments. The response of soybean was measured as visual injury ratings (at 7 and 28 days after treatment – DAT) as well as effects on yield components and yield. Broadcast flaming conducted once (at VC or V5 stage), as well as twice (at VC and V5 stages) exhibited the lowest injury of about 8% at 28 DAT. Any treatment that contained flaming at V2 stage resulted in more than 70% injury at 28 DAT. The highest crop yields were obtained from the non-flamed control (3.45 t ha⁻¹) and the plots flamed once at VC (3.35 t ha⁻¹), V5 (3.32 t ha⁻¹), and two times at VC and V5 (3.24 t ha⁻¹), which were all statistically similar. Soybean flamed at V2 stage had lower yields (1.03 t ha⁻¹ at V2, 0.46 t ha⁻¹ at VC and V2, and 0.38 t ha⁻¹ at V2 and V5). The lowest yields were in soybean flamed three times (VC, V2, and V5 stages), which yielded only 0.36 t ha⁻¹. These results indicate that soybean could tolerate a maximum of two flaming treatments at VC and V5 growth stages per season without any yield reduction.     

Validating the effectiveness of alternative euthanasia techniques using penetrating captive bolt guns in mature swine (Sus scrofa domesticus)

Euthanasia of mature swine is challenging. Temporal and behind-the-ear locations are two sites that have been identified as alternatives to the more commonly used frontal placement. In stage one, the effectiveness of two penetrating captive bolt gun styles (cylinder or pistol) was evaluated using frontal, temporal, and behind-the-ear placement in anesthetized mature swine (n = 36; weight: 267 ± 41 kg). For stage one, when evaluating treatment efficacy by sex, the cylinder-style equipment was 100% effective in achieving death when applied to all cranial locations (frontal, temporal, and behind-the-ear) for sows; however, the pistol-style equipment was only 100% effective when applied at the behind-the-ear location for sows. For boars, the cylinder-style equipment was 100% effective when applied to the frontal and behind-the-ear location, but the pistol-style equipment was not effective for any cranial location in boars. Therefore, the pistol–frontal, pistol–temporal, pistol–behind-the-ear, and cylinder–temporal were not included for boars, and pistol–frontal and pistol–temporal were not included for sows in stage two. In stage two, commercial, mixed-breed, mature swine (n = 42; weight: 292 +/− 56 kg) were randomly assigned to one of four treatments based on the inclusion criteria described in stage one. A three-point traumatic brain injury (TBI) score (0 = normal; 1 = some abnormalities; 2 = grossly abnormal, unrecognizable) was used to evaluate six neuroanatomical structures (cerebral cortex, cerebellum, hypothalamus, thalamus, pons, and brain stem), and the presence of hemorrhage was also noted. All treatments were 100% effective in stage two. A significant interaction between gun style and placement was determined on predicting total TBI as the cylinder style produced a higher total TBI score compared with the pistol type of the magnitude of +2.8 (P < 0.01). The cylinder style tended to produce a greater TBI score than the pistol in the temporal location (+1.2; P = 0.08). No difference was noted for TBI score behind-the-ear between the cylinder- and pistol-style gun (P > 0.05). TBI tended to be less in boars compared with sows (−0.6; P = 0.08). Hemorrhage was observed in frontal, parietal, occipital, and temporal lobes. This study demonstrated that the cylinder-style captive bolt gun more effectively resulted in brain trauma and death compared with a pistol-style gun and the behind-the-ear and temporal placement showed promise as an alternative placement site for euthanizing mature pigs on-farm.     

Setaria digitata was the main cause of equine neurological ataxia in Korea: 50 cases (2015–2016)

This study was performed to examine and clarify the cause of hindlimb ataxia and neuropathy seen in the South Korean horse population. Fifty horses diagnosed with hindlimb ataxia and neuropathy were referred for this study. Neurological examination was performed on 47 horses while necropsy was performed in all 50 animals. The occurrence of neurological diseases increased rapidly in the summer and 47 out of 50 horses were referred after the end of July. The incidence of neurological diseases started from the southern part of Korea in July and proceeded northward in August and September. Although there was no correlation with age, Thoroughbred and Warmblood horses showed a higher incidence rate than Halla and Jeju horses. The incidence rate was 5 times higher in geldings than in mares and stallions. Of the 20 cases, 16 were diagnosed with eosinophilic meningoencephalomyelitis in 2015. The most common lesions observed in 2016 were parasitic meningoencephalomyelitis (10 cases, 33%) and eosinophilic meningomyelitis (7 cases, 23%). Histopathological analysis of the brain and spinal cord revealed nematodes of approximately 100–200 µm in diameter, microcavitation and infiltrates of eosinophils, and brown pigmented macrophage infiltrates. The nematodes were identified as Setaria digitata via DNA sequencing, performed subsequent to polymerase chain reaction using DNA isolated from formalin-fixed paraffin-embedded tissue sections of the spinal cord. These results show that aberrant migration of Setaria digitata larva in the brain and spinal cord was a major cause for neurological signs in horses.     

施用有机肥对当归药材性状、产量及抗病性的影响

为了探寻有机肥对当归成药栽培的影响,采用黑膜覆盖栽培,基施纯有机肥（O,2000 kg·hm^-2）、纯化肥（C,磷酸二铵,420 kg·hm^-2）、减半量化肥增施有机肥［1/2（O+C）］,以不施肥为对照（CK）,栽培期测定早期抽薹率、发病率、药材产量和性状指标。结果表明,纯有机肥栽培当归早期抽薹率为8.0%,分别较CK、1/2（O+C）和C降低19.4%、15.2%和7.1%。药材产量则相反,O、C和1/2（O+C）栽培当归药材产量相当,鲜产量分别较CK提高105.9%、84.6%和78.2%。纯有机肥栽培当归根最长,侧根最多,单根最重,药材产量最高。化肥对根的增粗作用最大,但含水量高,根病率高,发病重。不同施肥每hm²鲜药材产量与综合评价指数大小顺序一致,依次为O（9220.6 kg,0.926）>C（9038.5 kg,0.610）>1/2（O+C）（8728.4 kg,0.481）>CK（4897.4 kg,0.190）。每hm²干药材产量排序为O（3149.2 kg）>1/2（O+C）（3098.7 kg）>C（2909.2 kg）>CK（1707.0 kg）,与经济收益一致。在岷县道地产区黑膜覆盖栽培,纯施有机肥对当归药材具有显著增效作用,可有效降低早薹率、根病率和发病程度,改善药材性状,提高药材产量和质量,在当归标准化栽培中可推广应用,以改变当归依赖化肥的生产现状,促进绿色有机栽培。     

三种荒漠植物叶绿素荧光参数日变化特征

利用Photosynq MultiseQ多功能植物测量仪测定塔克拉玛干沙漠腹地3种主要防护林植物头状沙拐枣、多枝柽柳和梭梭的叶绿素荧光参数日变化,探讨其与环境因子的关系,以揭示3种植物对极端环境的适应策略,为沙漠公路防护林的管护提供理论依据。结果表明：1）3种土壤都呈碱性;0~30 cm土层中的电导率（EC）远高于30 cm以下的土层。1 m内梭梭土层中的含水量高于多枝柽柳高于头状沙拐枣。2）3种植物的光合有效辐射（PAR）、叶片温度和线性电子传递（LEF）及植物周围的大气温度日变化均先升后降,且都在14：00达到最高;而大气湿度、实际光化学效率Y_（II）日变化均呈‘V’型。3）PAR基本上与3种植物的LEF、非光化学淬灭系数（NPQ）、调节性能量耗散量子产量Y_（NPQ）、非调节性能量耗散量子产量Y_（NO）呈显著正相关,与实际光化学效率Y_（II）呈显著负相关;3种植物的Y_（II）与Y_（NPQ）、Y_（NO） 均呈极显著负相关。3种植物的LEF、Y_（II）、Y_（NPQ）的日均值差异不显著,但梭梭与多枝柽柳的NPQ和Y_（NO）日均值差异显著。4）在3种植物的叶绿素荧光参数中,梭梭和头状沙拐枣的LEF、NPQ和Y_（II）要高于多枝柽柳;而多枝柽柳的Y_（NO）要显著高于头状沙拐枣和梭梭。因此,高温和高光均对3种植物造成了不同程度的影响,通过主成分分析表明,3种植物的抗逆性强弱顺序分别为梭梭>头状沙拐枣>多枝柽柳。     

1990—2019年阿拉尔垦区植被覆盖时空变化特征分析

监测植被覆盖变化对区域生态环境评价和可持续发展具有重要意义。本文基于Landsat遥感影像和相关统计数据,运用像元二分模型和重心转移模型等方法分析阿拉尔垦区1990—2019年近30年的植被覆盖时空变化特征。结果表明：1990—2019年,阿拉尔垦区平均植被覆盖面积呈增加趋势,高和极高植被覆盖区的面积呈增加趋势,差异显著,分别增加了674 km²和1 147 km²;阿拉尔垦区植被覆盖变化存在时段性和区域性差异,时段上,2005—2010年垦区西北部开始出现植被,以中覆盖类型为主,区域上,垦区西北部植被覆盖的增加趋势最显著;近30年,垦区植被覆盖重心整体向东北方向转移;气候对阿拉尔垦区植被覆盖变化有一定的影响,但人类活动影响最直接。在未来发展中应注重保护自然植被,有计划地开垦土地,合理调整垦区农业结构,科学分配水权,保证垦区生态用水,实现可持续发展。     

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.     

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.