Mutations in the US2 and glycoprotein B genes of the equine herpesvirus 1 vaccine strain RacH have no effects on its attenuation]

The equine herpesvirus 1 (EHV-1) modified live vaccine strain RacH is apathogenic for both laboratory animals and the natural host. The apathogenicity of RacH was caused by serial passages of the virus in heterologous cells. When compared to the virulent parental strain RacL11 several changes in the RacH genome occurred. Previous results have shown that the loss of the IR6 gene correlated with the loss of virulence. Additional important mutations were observed within the US2 gene which is directly adjacent to the IR6 gene and within the glycoprotein B (gB) gene. To answer the question whether these mutations contribute to the attenuation of RacH several recombinant EHV-1 were constructed: The mutated genes in RacH were replaced by the wild-type US2 gene or the wild-type gB gene, respectively. In addition, a RacL11 recombinant expressing the mutated (RacH) gB instead of the wild-type gene was generated. All recombinant viruses were tested for virulence using the EHV-1 mouse model. The results were as follows: i) The insertion of the RacL11 US2 gene into the RacH virus did not restore virulence and none of the infected mice showed typical signs of EHV-1-caused disease (symptoms and body weight loss). ii) Exchanging gB genes between RacL11 and RacH did not alter their virulence phenotypes remarkably either. Therefore, it is concluded that attenuation of the EHV-1 vaccine strain RacH is caused solely by the absence of the IR6 gene and protein.     

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.     

Equine herpesvirus type-1 modulates CCL2, CCL3, CCL5, CXCL9, and CXCL10 chemokine expression

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential.     

Cloning of the Genomes of Equine Herpesvirus Type 1 (EHV‐1) Strains KyA and RacL11 as Bacterial Artificial Chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV‐1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Escherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co‐transfection of viral DNA and recombinant plasmid pΔ71‐pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high‐molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full‐length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacL11 and pKyA, respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP‐expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full‐length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2‐negative progeny reconstituted from pRacL11 and pKyA (L11Δgp2 and KyAΔgp2) exhibited different growth properties. Whereas both L11Δgp2 and KyAΔgp2 extracellular titres were reduced by 15‐ to 32‐fold, plaque diameters were only markedly (50%) reduced in the case of KyAΔgp2.     

Parasites in lungs of dead equids in Kentucky: emphasis on Dictyocaulus arnfieldi

Prevalence of natural infections of parasites from the lungs of 488 dead Thoroughbreds in Kentucky was investigated. The horses varied from 1 to 32 years of age; 419 horses were from 215 farms and 69 horses were from 68 individual sources for which a specific farm was not identified. Examinations of the lungs were made from Mar 1, 1983 through Feb 29, 1984. Dictyocaulus arnfieldi was recovered from 56 (11%) of the horses. Other parasites found were larvae of Parascaris equorum in 37 (8%) and of Habronema/Draschia in 67 (14%) of the horses. The possible effect of ivermectin treatment on the prevalence of D arnfieldi in the lungs is discussed. Presence of D arnfieldi in 20 other selected dead equids was also investigated.     

3个草地早熟禾品种分蘖期的生理特性变化

以草地早熟禾午夜2号、巴润和肯塔基为材料,测定其分蘖期生理指标的变化。结果表明：午夜2号、肯塔基和巴润叶绿素和可溶性蛋白含量变化趋势一致,呈单峰曲线,在较高水平上峰值分别为叶绿素含量2.215,2.000和2.013mg/g;可溶性蛋白0.85,0.64和0.77mg/g。当维持一段时间后开始下降;而3个草地早熟品种可溶性糖含量与过氧化氢酶活性随着生长时间的延长而升高;在分蘖过程中,3个草地早熟禾品种平均分蘖数随着生长时间的延长呈单峰曲线,在发芽后第35d午夜2号2.45,巴润和肯塔基1.98分蘖枝/株达到最大值。草地早熟禾分蘖期间,叶绿素、可溶性蛋白含量与平均分蘖数呈正相关,而可溶性糖与过氧化氢酶与平均分蘖数相关性不明显。同一生长期内,午夜2号叶绿素含量、可溶性蛋白、可溶性糖、过氧化氢酶含量以及平均分蘖数均大于巴润和肯塔基,肯塔基含量最低。     

紫花苜蓿与草地早熟禾轮作模式对其农艺性状和营养品质的影响

为研究紫花苜蓿（Medicago sativa,本试验中记为A）与草地早熟禾（Poa pratensis,本试验中记为K）轮作对其农艺性状和营养品质的影响,本试验以种植5年的紫花苜蓿（A）和草地早熟禾（K）为前茬,在2块草地上分别种植紫花苜蓿（A）和草地早熟禾（K）,即形成AA,AK,KA,KK 4种种植模式,以AK和KA为轮作处理,AA和KK为连作对照。研究了轮作对紫花苜蓿和草地早熟禾各刈割茬次的株高、干草产量、干鲜比、茎叶比、粗蛋白（Crude protein,CP）、中性洗涤纤维（Neutral detergent fiber,NDF）和酸性洗涤纤维（Acid detergent fiber,ADF）等指标变化的影响。结果表明：第1~3茬,AK轮作草地早熟禾的株高较KK连作提高6.31%~9.47%,且在第2茬差异显著（P<0.05）。KA轮作紫花苜蓿的株高较AA连作提高6.72%~9.02%,且在第2茬差异显著（P<0.05）。AK轮作的草地早熟禾年干草产量较KK连作提高8.54%,KA轮作的紫花苜蓿年干草产量较AA连作提高11.57%。AK轮作草地早熟禾的干鲜比较KK连作在3茬间差异显著（P<0.05）,且增加幅度大于KA比AA的增加幅度。不同轮作模式对茎叶比的提高在后2茬差异明显。AK轮作下,草地早熟禾的粗蛋白含量比KK提高14.16%~15.95%,增长幅度优于KA比AA的增加幅度。AK轮作和KA轮作的中性洗涤纤维含量和酸性洗涤纤维在不同茬次较KK连作和AA连作模式差异显著（P<0.05）。紫花苜蓿与草地早熟禾轮作可以提高后茬牧草的株高、干草产量、茎叶比和干鲜比以及粗蛋白含量;并且降低后茬的酸性洗涤纤维和中性洗涤纤维。本试验旨在通过探究紫花苜蓿与草地早熟禾轮作下牧草农艺性状和营养成分的变化情况,为今后豆-禾牧草轮作、草种搭配以及模式排布提供借鉴依据。     

Eyeworms (Thelazia lacrymalis) in one- to four-year-old Thoroughbreds at necropsy in Kentucky (1984 to 1985)

From Mar 1, 1984, to Feb 28, 1985, both eyes from 189 one- to four-year-old Thoroughbreds (88 males, 90 females, and 11 geldings) in Kentucky were examined at necropsy for eyeworms. Thelazia lacrymalis was recovered from 45% of 1-year-old horses, 26% of 2-year-old horses, 45% of 3-year-old horses, and 50% of 4-year-old horses; overall, 43% of 1- to 4-year old horses were infected. Prevalence of eyeworms in horses in the present study was comparable with that in horses of similar ages examined at necropsy in Kentucky in 2 previous studies (1975 to 1976 and in 1979), except for the lower percentage of infected 2-year-old Thoroughbreds in the present study.     

不同早熟禾品种对干旱胁迫的生理响应及抗旱性评价

为鉴定评价早熟禾幼苗抗旱性,将14个早熟禾品种的种子经消毒后播种在塑料花盆中,出苗后间苗,2~3个真叶定株,每盆留生长一致、分布均匀的幼苗15株,定期浇水,以保证幼苗的正常生长。待生长到3~4个真叶时进行干旱处理,依据Michel公式计算不同水势所需的PEG-6000的量,然后加入PEG-6000溶液进行干旱处理,处理的水势梯度为0 MPa(即为对照),-0.3,-0.6,-0.9和-1.2 MPa。处理后每2 d进行称重补水以维持溶液浓度。设干旱处理与对照(正常浇水)两组,对14个早熟禾品种幼苗进行模拟干旱胁迫,4次重复。测定叶绿素(CHL)含量、丙二醛(MDA)含量、脯氨酸(Pro)含量、过氧化物酶(POD)以及超氧化物歧化酶(SOD)活性等生理生化指标。结果表明,随着干旱胁迫的加剧,早熟禾幼苗叶片CHL含量呈下降趋势;Pro含量、MDA含量、SOD活性总体呈上升趋势;POD活性总体呈先降再升后降的趋势;利用隶属函数分析得出14个早熟禾品种的抗旱性强弱顺序依次为:3号恩托佩草地早熟禾>10号细叶早熟禾>1号优异草地早熟禾>2号斗士草地早熟禾=9号蓝钻草地早熟禾>7号草地早熟禾>11号草地早熟禾>4号草地早熟禾>5号瓦巴斯草地早熟禾>8号早熟禾=13号肯塔基草地早熟禾>14号草地早熟禾>6号莫诺波利草地早熟禾=12号菲尔津草地早熟禾。     

Deletion of gene 52 encoding glycoprotein M of equine herpesvirus type 1 strain RacH results in increased immunogenicity

The immunogenicity of equine herpesvirus type 1 (EHV-1) strain RacH was compared to a RacH virus in which gene 52 encoding glycoprotein M (gM) was interrupted by insertion of LacZ (HDeltagM-Ins) and a RacH with 75% of gene 52 was deleted and replaced by LacZ (HDeltagM-HS). HDeltagM-Ins failed to produce full-length gM, but the carboxy-terminal portion was still expressed. No gM expression was detected in HDeltagM-HS-infected cells. Mice were immunised once with 1x10(3) to 1x10(5) plaque-forming units (PFU) of RacH or mutant viruses and challenged with virulent RacL11 virus 29 days later. A dose-dependence of protection was observed in RacH-immunised mice, and following immunisation with 1x10(4) or 1x10(3) PFU body weight losses and increased virus titres in lungs were observed after challenge infection. HDeltagM-HS-immunised mice were completely protected even after immunisation with 1x10(3) PFU. Mice immunised with 1x10(3) PFU of HDeltagM-Ins but not the higher doses showed signs of disease after challenge infection.     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

NaCl胁迫对15个草地早熟禾品种萌发的影响

对15个草地早熟禾品种（系）种子在0、0．2％、0．4％、0．8％、1．0％浓度NaCl胁迫下的发芽率、发芽势、相对盐害率等指标进行了测定,结果表明,15个草地早熟禾品种（系）的发芽率、发芽势都随着NaCl浓度的递增而下降,相对盐害率则随着NaCl浓度递增而增加,表明盐胁迫对草地早熟禾种子的萌发有明显的抑制作用。对各指标综合评价后筛选出耐盐材料2份031315和031322草地早熟禾;敏盐材料3份031310,031312和031318草地早熟禾,均来自黑龙江省,其他为中等耐盐材料。     

草地早熟禾转CMO-BADH双基因和转CMO基因耐盐性分析

通过对非转基因草地早熟禾(Poa pratensis L.)、转CMO-BADH双基因草地早熟禾、转CMO基因草地早熟禾进行不同浓度的NaCl胁迫试验,测定其细胞膜的透性,丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、氧化氢酶(CAT)活性,评定各株系耐盐能力的强弱,同时还验证并比较了CMO-BADH双基因和CMO基因的耐盐性功能,为耐盐新品种的选育提供了理论依据。结果表明:所有株系的相对电导率、MDA含量均随盐浓度增加而增大,在NaCl胁迫下相对电导率和MDA含量的大小顺序为:非转基因株系>转CMO基因株系>转CMO-BADH双基因株系;在NaCl的胁迫下,各个株系的SOD活性、POD活性、CAT活性都有明显的提高,此3种指标的大小顺序为:转CMO-BADH双基因株系>转CMO基因株系>非转基因株系;综合考虑各个指标,各株系耐盐性强弱顺序为:转CMO-BADH双基因株系>转CMO基因株系>非转基因株系。     

灌溉对草地早熟禾坪用质量的影响

应用水量平衡原理,通过线性梯度灌溉,控制对草坪草的灌溉量,研究新疆地区不同水分处理对草地早熟禾（Poa pratensis）草坪密度、色泽、质地、均一度的影响。结果表明,草地早熟禾草坪在B3（田间最大持水量的60%～70%）水分处理下可以得到较好的色泽、均一性和密度,但草地早熟禾质地评分在不同水分处理间差异不大,表明水分不能有效影响草地早熟禾质地。通过对不同水分处理下的草地早熟禾草坪质量进行综合评分,经方差分析发现草地早熟禾草坪在B3水分处理下就可以满足其生长需求,达到较好的外观质量。     

不同草坪草冠层反射光谱特征的比较研究

选用高羊茅、多年生黑麦草、草地早熟禾、狗牙根、匍匐翦股颖5种草坪草的24个品种为试验对象,测定冠层反射光谱,比较可见光和近红外区域反射率的差异性,分析多种红边参数与草坪色泽目测评分值之间的相关性。结果表明,高羊茅8个品种在450～900 nm的反射率无显著性差异;草地早熟禾5个品种可见光450～650 nm的反射率存在差异显著性(P<0.05);多年生黑麦草8个品种在近红外750～900 nm的反射率存在差异显著性(P<0.01)。高羊茅美洲虎、多年生黑麦草威士、草地早熟禾蓝宝石、狗牙根太阳城、匍匐翦股颖普特在550、565、8008、50 nm处均表现出差异显著性(P<0.05),红边斜率与草坪色泽目测评分值之间存在显著相关关系。利用高光谱分析技术可以快速、准确地反演草坪冠层叶绿素含量,并且能够区分同一草坪草种品种间的反射率差异。     

草地早熟禾的石蜡切片制作技术的优化

以草地早熟禾为材料,在传统石蜡切片方法的基础上,结合草地早熟禾组织结构特点,对脱水、透明、染色等重要制片环节进行改良。结果显示:脱水过程中,将乙醇逐级梯度脱水时间增加,有利于后续浸蜡,避免石蜡切片上的植物材料脱落;透明过程中,将纯二甲苯增加更换频率,同时减少每次处理时间,其透明更彻底;有效的番红固绿染色时间组合处理,使草地早熟禾解剖形态更为清晰。一系列针对草地早熟禾石蜡切片的优化,使得解剖图像染色均匀、图像清晰,不易出现植物材料脱落及组织结构不完整的情况,是观察草地早熟禾形态结构的有效方法。     

Horse Preference,Forage Yield,and Species Persistence of 12 Perennial Cool-Season Grass Mixtures Under Horse Grazing

Cool-season grass mixtures are rarely evaluated for preference, yield, and persistence under horse grazing. The objectives of this research were to evaluate horse preference, forage yield, and persistence of cool-season grass mixtures under horse grazing. Eight commercially marketed and four experimental perennial cool-season grass mixtures were planted in 2009 in a randomized complete block with five replicates and grazed by four adult horses during 2010, 2011, and 2012. All mixtures contained four to six cool-season perennial grass species. Specie density measurements were taken in each spring and fall, and yield was mechanically measured before each grazing period. After grazing, preference was determined by visually assessing percentage of forage removal on a scale of 0 (no grazing) to 100 (100% of vegetation removed). Data were analyzed using a mixed-model analysis of variance and liner regression. Horses preferred mixtures containing tall fescue, perennial ryegrass, Kentucky bluegrass, and timothy (P < .001). Horses had less preference for mixtures containing ≥30% orchardgrass (P < .001). Mixtures had similar (P = .11) forage yields that ranged from 6,100 to 7,082 kg ha⁻¹. After 2 years of grazing, orchardgrass and tall fescue increased; Kentucky bluegrass remained stable; and festulolium, meadow fescue, and perennial ryegrass had the greatest rate of decline in mixtures. Orchardgrass became the dominate species, regardless of initial percentage in the mixture. Mixtures containing tall fescue, perennial ryegrass, Kentucky bluegrass, and timothy should be planted in midwestern US horse pastures; however, mixtures will likely transition to tall fescue and Kentucky bluegrass–dominated pastures.     

Effect of Water Extractions from the Leaves and Rhizomes of Kentucky Bluegrass on Seed Germination of Three Herbaceous Ornamentals北大核心CSCD

In order to explore the synergistic growth of Kentucky bluegrass and herbaceous ornamentals in the garden plants configuration, the water extractions from leaves and rhizomes of Kentucky bluegrass (Poa pratensis) was used to examine the allelopathic effects on seed germination of Orychophragmus violaceus Zinnia elegans Tagetes patula using petridishs with filter papers. Results showed that the water extractions of the leaves and rhizomes of Kentucky bluegrass inhibited seed germination of Orychophragmus violaceus, Zinnia elegans, and Tagetes patula by 33% and 48%, 27% and 39 %, and 24% and 48%, respectively. And the rhizome extraction appeared to be more effective than leaf extraction in terms of inhibiting the seed germination, and the growth of radicle and coleoptile. Three ornamentals also showed different sensitivity to the extractions of Kentucky bluegrass, and Orychophragmus was the most sensitive to the extractions. This study showed that Orychophragmus should not be planted with Kentucky bluegrass to avoid alleloathic effect on growth by Kentucky bluegrass. © 2019 China Agricultural University. All rights reserved.     

2,4-D和6-BA对早熟禾愈伤组织诱导的影响

早熟禾愈伤组织的诱导是建立早熟禾高频再生体系的前提和基础。本试验以早熟禾成熟种子作为外植体,MS为基本培养基,在不同的2,4 D和6 BA浓度下诱导愈伤组织。结果表明早熟禾不同品种间,愈伤组织诱导率显著不同。在2,4 D和6 BA配合使用的情况下,愈伤组织的质量和诱导率要优于单独使用2,4 D的情况。     

Salmonella enterica isolated from wildlife at two Ohio rehabilitation centers.

Between May and September 2004, fecal samples from various wildlife species admitted to two rehabilitation centers in Ohio were cultured for Salmonella enterica and Escherichia coli O157:H7. Eight of 71 (11%) samples, including specimens from three opossums (Didelphis virginiana), two gray squirrels (Sciurus carolinensis), a woodchuck (Marmota monax), a Harris hawk (Parabuteo unicinctus), and a screech owl (Otus asio) tested positive for Salmonella serovars Braenderup, Senftenberg, Oranienburg, and Kentucky. The Salmonella Oranienburg isolates were indistinguishable by pulsed-field gel electrophoresis. Most isolates were susceptible to commonly used antibiotics; however, the Salmonella Kentucky isolate was resistant to multiple beta-lactam antibiotics (amoxicillin/clavulanic acid and ampicillin), cefoxitin, and ceftiofur, a third-generation cephalosporin. Escherichia coli O157:H7 was not isolated from any sample. Transmission of Salmonella from wildlife may occur between animals at rehabilitation centers.