Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) for Identification of Bacterial Isolates From Horses

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is used for bacterial identification by analyzing the spectra of isolates and comparing them against a database of reference spectra; it is known for its rapidity and accuracy. Although MALDI-TOF MS is used for identification of bacterial isolates from animals, not all animal pathogens are identified correctly. In this study, we used a commercial MALDI-TOF MS identification system to examine 3724 bacterial isolates from horses and their environments. Isolates that could not be identified with MALDI-TOF MS were identified by 16S rRNA gene sequence taxonomic analysis. MALDI-TOF MS could identify 86.2% of the isolates from horses to the species level, showing that this method could be successfully applied for bacterial identification in horses. However, some species known to be equine pathogenic agents including Taylorella equigenitalis and Rhodococcus equi were difficult to identify with MALDI-TOF MS, which might be the result of an inadequate reference database. Some Prevotella, Staphylococcus, and Streptococcus isolates, which could not be identified with either MALDI-TOF MS or 16S rRNA gene sequencing analysis, formed clusters in the 16S rRNA phylogenic tree, and might be unknown species isolated from horses. Adding the spectra of isolates identified in this study to an in-house database might make MALDI-TOF MS a more useful tool for identifying equine isolates.     

Rapid Identification of Mycoplasma pulmonis Isolated from Laboratory Mice and Rats Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.     

基质辅助激光解析电离飞行时间质谱在动物病原菌检测中的应用

应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）技术,对厦门口岸进口的1000多份动物及其产品的样品进行致病菌的检测和鉴定,共检出20多种致病菌,并对这些菌株同时用传统生化鉴定方法进行确认。结果表明MALDI-TOF-MS对未知细菌进行鉴定,较传统方法更加快速、准确,而且可以进行高通量检测,可以广泛应用于口岸动物检疫以及微生物检验实验室的日常检验。此外,将国内分离鉴定的各种参考菌株建立的常见致病菌MALDI-TOF-MS数据库与布鲁克公司的MALDI Biotyper数据库进行病原菌鉴定的比较,结果表明,实验室自建的MALDI-TOF-MS数据库可以取得更加准确地结果。     

基质辅助激光解吸电离飞行时间质谱在动物源细菌耐药性监测中的研究应用

细菌鉴定是细菌耐药性监测过程中的重要工作环节之一,基质辅助激光解析电离飞行时间质谱(Matrix-assisted laser desorption ionization time-off flight mass spectrometry, MALDI-TOF MS)能够高效鉴定细菌。为了快速监测五家养殖场来源的大肠杆菌和肠球菌的临床耐药特征,本研究利用MALDI-TOF MS和微量肉汤稀释法,快速鉴定临床分离的大肠杆菌和肠球菌,并对其进行耐药表型检测。结果显示,MALDI-TOF MS实现了对临床分离菌株(31株大肠杆菌和34株肠球菌)的快速鉴定;鸡源大肠杆菌和肠球菌的耐药情况最为严重,其次为羊和牛。其中,鸡源的大肠杆菌均对四环素(100%)和氨苄西林(91.67%)耐药率最高,肠球菌对苯唑西林(62.07%)耐药率较高。研究结果表明,不同动物源细菌临床耐药性表型严重程度有所不同,与此同时,MALDI-TOF MS技术可以同时实现对动物源大肠杆菌和肠球菌的快速鉴定,值得在动物源细菌耐药性检测领域推广应用。     

飞行时间质谱法检测水牛黑素皮质素受体4基因多态性

应用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF-MS)检测水牛黑素皮质素受体4(MC4R)基因的多态性(SNPs),为今后建立水牛辅助标记选择策略奠定基础。以水牛MC4R基因序列为模板,利用PCR扩增测序法筛查了水牛MC4R基因13个SNPs。为进一步验证筛选的可靠性,应用MALDI-TOF-MS针对380头水牛检测了8个标记的基因型。结果显示,平均检出率为98.0%,能够准确的检测出3种常见的基因型。所检SNP位点的平均最小等位基因频率(MAF)为0.24,平均杂合度为0.28,平均多态信息含量(PIC)为0.23。本研究建立的基于PCR测序法联合MALDI-TOF-MS检测水牛MC4R基因多态性的方法,具有快速、可靠和准确的特点,为今后开展水牛生产性状基因的SNP分型和检测等研究奠定了基础。     

Rapid Detection of SNPs in Buffalo MC4R Gene by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The study used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for rapid determination of single nucleotide polymorphisms (SNPs) in buffalo MC4R gene,which provided a foundation for constructing the marker-assisted selection(MAS)strategy.The cDNA sequence of buffalo MC4R gene was screened rapidly based on PCR amplification and sequencing method,and in which 13 SNPs were found.To further identify the reliability of screening system,8 SNPs were genotyped in 380 buffaloes using the MALDI-TOF-MS technology.The results revealed that 8 SNPs had an average of 98.0% in call rate,and in which three genotypes could be accurately distinguished.The average minor allele frequency (MAF),average heterozygosity and polymorphism information content (PIC) value of the 8 markers was 0.24,0.28 and 0.23,respectively.Our findings indicated that the rapid determination method of MALDI-TOF-MS with PCR and sequencing with highly accurate properties was a useful tool for identifying the SNPs of buffalo MC4R gene that laid the foundations for studying SNP genotype in buffalo genes associated production traits.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

The Use of Sex-Sorted Stallion Semen in Embryo Transfer Programs

Sorting stallion semen into two separate populations of enriched X- or Y-bearing sperm can be done successfully. For this, stallion semen can be shipped to a sorting facility, but the mare must be in close to the sorting laboratory. Fertility rates when using 20-40 million sperm are an acceptable 60% per insemination. The procedure can be implemented in embryo transfer programs, with no deleterious effect on the pregnancy rate or embryonic death.     

Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.     

飞行时间质谱分析技术及其应用研究进展

阐述了飞行时间质谱分析技术的基本原理、仪器构成、性能特点,总结了飞行时间质谱分析技术的应用进展,并对这一技术的应用前景进行了展望。     

马冷冻精液氧自由基的产生和清除系统

马精液中存在相对稳定的氧自由基(ROS)产生和清除系统,当精液经离心去除精清后,稳定的系统遭到破坏。马精液冷冻和解冻过程不能有效的清除ROS,将导致解冻后精子各项生理功能的下降,直接影响冻精的质量,而添加外源性的抗氧化剂则可以重新使精液的ROS产生和清除达到平衡。作者综述了马精液中精子细胞、白细胞ROS的产生系统和精液GSH-PX、CAT、SOD、VA、VE等对ROS的清除系统,旨在对人们研究马精液冷冻保存时精子中ROS的损伤机理研究提供一定的帮助。     

家蚕蚁蚕蛋白质组的质谱鉴定与数据库构建

为尝试构建基于质谱分析的家蚕蛋白质组数据库,采用一维电泳-液相色谱-质谱(1DE-LC-MS)技术对家蚕蚁蚕的蛋白质组进行了分析与鉴定。共检索到511个相匹配的候选蛋白质和92个多肽离子片段,鉴定获得了171个无冗余蛋白质组分,分别属于121种蛋白质或蛋白质亚基,其中有36种蛋白质或亚基尚未在家蚕及昆虫的基因与蛋白质数据库中报道;在171个被鉴定的蛋白质组分中,与细胞骨架和蛋白质代谢相关的组分多达64种。结果还表明,1DE-LC-MS技术也适用于从一维凝胶电泳的差异性条带及蛋白质混合样品中分离和鉴定特异蛋白质组分。     

大豆卵磷脂对马精液冷冻保存的效果研究

为探索适宜浓度的大豆卵磷脂(soybean lecithin,SL)代替卵黄(egg yolk,EY)对马精液冷冻保存的效果,本试验分别以5%卵黄(V/V)和10%、20%、30%大豆卵磷脂(m/V)作为精液的冷冻保护剂冷冻解冻马精液,解冻后分别对精子细胞的运动参数、精子细胞膜的完整性、精子细胞脂质氧化物丙二醛的值和线粒体膜完整性进行检测。结果发现,精液冷冻解冻后,5%卵黄和10%、20%、30%大豆卵磷脂对精子的总运动精子数无显著影响(P>0.05),但30%大豆卵磷脂具有最大的原地摆动精子数和最小的直线前进精子数(P<0.05),其他运动参数差异不显著(P>0.05);20%大豆卵磷脂代替卵黄后具有最高的质膜完整性,同时产生最少的脂质氧化物丙二醛;线粒体膜电位经流式细胞仪检测时,30%大豆卵磷脂活精子高膜电位数最高,但与20%大豆卵磷脂之间差异不显著(P>0.05)。试验结果表明,20%大豆卵磷脂能够代替卵黄作为冷冻保护剂用于马精液的冷冻保存。     

Colloidal Centrifugation of Stallion Semen: Changes in Sperm Motility,Velocity, and Chromatin Integrity during Storage

The current study investigated the changes in sperm quality (motility, velocity, and chromatin integrity) occurring during storage at room temperature or 5°C for up to 48 hours in spermatozoa after extension or single-layer centrifugation (SLC) through Androcoll-E. In unselected samples, all parameters of sperm quality deteriorated significantly during storage (P < .01), although the deterioration was faster at room temperature (22–30°C) than for cool storage (P < .01). The SLC-selected spermatozoa had higher motility, velocity, and chromatin integrity than the overall unselected population (motility: selected 85 ± 10%, unselected 56 ± 13%; P < .001; velocity: selected 85.1 ± 13 μm/second, unselected 63.5 ± 15 μm/second; P < .001; and DFI selected 12.2 ± 4.8 μm/second, unselected 23.6 ± 7.4 μm/second; P < .001). Furthermore, sperm quality did not deteriorate with storage in the SLC-selected samples, either at room temperature (22–30°C for 24 hours) or cooled to 4°C (for at least 48 hours), whereas a significant deterioration in sperm quality was observed in the unselected sperm samples (P < .01). Thus, room temperature storage of SLC-selected spermatozoa may be an option for insemination doses from stallions whose spermatozoa do not tolerate cooling. In addition, a new sperm analyzer, the Qualisperm, showed good correlation with subjective motility assessment (r = 0.8, P < .001), was user-friendly, and provided a reasonable volume of data. This instrument may be a useful adjunct to sperm quality assessment at the stud.     

Endoscopic Laser Ablation of a Midline Cyst of the Colliculus Seminalis as a Treatment for Anejaculation in a Stallion

An 18-year-old Appaloosa stallion presented with a history of ejaculatory dysfunction, which had recently progressed to an inability to ejaculate (anejaculation). Transrectal ultrasound evaluation revealed the presence of a prominent midline cyst of the colliculus seminalis, which was compressing the most terminal parts of the deferent ducts. Both ducts were enlarged and filled with hyperechoic content. The stallion was diagnosed with a complete occlusion of the deferent ducts because of the compression from the midline cyst of the colliculus seminalis. To date, there are no established treatments for this condition in stallions, although several procedures have been successfully used in men affected by similar problems. Therefore, we proposed performing one of these procedures—a laser ablation of the cyst to collapse it and to open the ejaculatory path. The stallion was placed under general anesthesia, and an endoscopic ultrasound-guided laser ablation of the cyst was performed. No immediate or long-term complications were observed. After the expulsion of accumulated material, the stallion produced normal semen and successfully impregnated mares during the next breeding season.     

超高压液相色谱-串联四级杆飞行时间质谱法同时筛查猪肉中54种兽药

应用超高压液相色谱串联四级杆飞行时间质谱建立了筛查猪肉中54种兽药的新方法。样品经过乙腈∶水＝5∶1（ V ∶ V）和5％三氯乙酸两次提取,旋蒸后经Waters Oasis HLB柱净化后上机测定,样品在正负离子模式下进行检测,在50～1500 Da质量范围内进行一级全扫描,以保留时间、化合物精确分子离子质量和特征碎片离子质量对药物进行定性分析。结果表明,在低中高三个添加浓度下测定了各种药物的回收率,54种兽药在4～1．5×104μg／L范围内线性关系良好,相关系数r2≥0．98。54种药物的质量数误差绝对值小于7．71×10-6,不同兽药在猪肉中回收率为10％～128％。猪肉中检出限为2～10μg／kg。该方法适用于猪肉中54种兽药的检测需要,该方法操作简单,准确,杂质干扰少。     

牛冷冻精液的应用研究进展

牛人工授精技术是所有家畜中应用最为广泛的一种繁殖技术,而其巨大的发展又得益于精液冷冻保存的成功应用.本文简要介绍了牛精液冷冻技术的发展历史和精液冷冻的原理,分析了影响牛精液冷冻效果的主要因素,并对国内外现行使用的精液评定指标作了简单阐述,以期为牛高质量冷冻精液的研究和牛人工授精技术的应用提供参考.     

基于飞行时间质谱法的单核细胞增生李斯特氏菌定性质控样品研制

通过真空冷冻干燥技术成功制备出单核细胞增生李斯特氏菌定性质控样品,并系统分析质控样品的均匀性和稳定性。通过优化冻干基质条件,得到制备质控样品的最佳条件。通过培养计数和基质辅助激光解吸电离飞行时间质谱技术,验证定性质控样品的均匀性和稳定性。结果表明：制备的单增李斯特菌定性质控样品为白色、质地均匀小球,均匀性验证实验质控样品培养计数结果F=0.567,小于临界值,表明均匀性一致;运输稳定性实验验证了质控样品在37、25?℃环境下含量几乎没有下降,计数稳定;贮藏稳定性实验验证了质控样品在－20?℃贮藏28?d后的复苏率为101.5%,在4?℃条件下贮藏28?d后的复苏率为99.6%,说明该样品均匀性和稳定性良好,可以作为阳性质控样品用于单增李斯特菌检测和质量控制。     

亲水作用色谱－电喷雾串联质谱法测定生鲜牛乳及奶粉中的双氰胺

建立了检测生鲜牛乳及奶粉中双氰胺的亲水作用色谱-电喷雾串联质谱(HILIC-ESI-MS/MS)分析方法.样品经乙腈提取后经亲水作用色谱柱分离,电喷雾串联四级杆质谱检测.结果显示,双氰胺标准曲线在0.5～100μg/L浓度范围内线性良好.相关系数(r)大于0.99;在5～50μg/kg添加水平下,回收率在80.1％～88.2％之间,相对标准偏差(RSD)均小于10％(n=6);方法操作简便、灵敏度高,适用于生鲜牛乳及奶粉中双氰胺的筛查及确证分析.