鸡毒支原体红霉素和替米考星耐药体外诱导及其23S rRNA基因V域突变特征分析

鸡毒支原体（Mycoplasma gallisepticum，MG）是禽类主要病原菌之一，其感染引起鸡慢性呼吸道病（CRD）和火鸡传染性窦炎。目前预防鸡毒支原体感染仍主要依靠抗菌药物，但长期广泛应用抗菌药物造成鸡毒支原体耐药性不断发展。有调查表明，鸡毒支原体临床株对四环素类、大环内酯类及氟喹诺酮类等抗菌药物已产生了耐药性，造成药物疗效下降甚至失效。由于支原体分离培养困难，国内外对动物源支原体耐药性研究十分有限，至今未见有鸡毒支原体大环内酯类抗生素耐药机制的研究报道。本文以鸡毒支原体S6株为模式菌，研究红霉素和替米考星在体外诱导鸡毒支原体的耐药情况，并分析耐药菌株23S rRNA基因V域的耐药突变特征。     

3种禽源支原体替米考星耐药株的体外诱导及23SrRNA基因V域碱基突变分析

作者拟探讨禽源支原体对替米考星耐药的分子机制.体外诱导得到鸡毒支原体(R株、PG31株和S6株)、鸡滑液支原体、衣阿华支原体的替米考星耐药株;PCR扩增原始敏感株和诱导耐药株的23S rRNA基因V域,测序分析耐药相关碱基突变情况.结果鸡毒支原体R株、PG31株、S6株、鸡滑液支原体、衣阿华支原体分别通过9代、8代、6代、14代、9代诱导获得替米考星耐药(≥128 μg·mL-1)株;3种禽源支原体替米考星诱导耐药株均对大环内酯类药物表现交叉耐药,23S rRNA基因发生A2503T突变的诱导耐药株对截短侧耳素类、氯霉素类药物的敏感性明显降低.诱导获得的替米考星耐药鸡毒支原体R株发生了A2058G和A2503T突变,PG31株发生了A2058G和A2059G突变,S6株发生了A2058G和A2503T突变;而诱导获得的替米考星耐药鸡滑液支原体发生了G2162A突变,衣阿华支原体发生了A2059C突变.本研究表明鸡毒支原体和衣阿华支原体在体外较易经替米考星诱导产生耐药性,而鸡滑液支原体相对较难.菌株23S rRNA基因V域2 058、2 059、2 503位点的碱基突变与替米考星耐药表型有密切关系.     

1株鸡毒支原体的分离鉴定及体外敏感中药的筛选

本研究旨在对山东泰安地区鸡毒支原体的流行菌株进行分离培养及鉴定和纯化,并针对该分离株筛选体外敏感的中药,为后续进一步研究提供材料。采用液体培养法与固体培养法从病鸡组织样品中分离、培养和纯化鸡毒支原体,并通过瑞士染色和姬姆萨染色、血清学方法及分子生物学方法对分离株进行初步鉴定,在此基础上,采用微量稀释法测定8种中药对分离菌株的最小抑菌浓度（MIC）,从而筛选到对分离菌株敏感的中药。结果显示,分离菌株在鸡毒支原体液体培养基中增殖后,培养基颜色由红变黄且呈半透明状态,在固体培养基中培养后呈典型的"煎蛋"样儿菌落,均符合鸡毒支原体培养特性;瑞士染色和姬姆萨染色结果显示,菌体形态符合鸡毒支原体的特征;血清学鉴定结果显示,分离株与鸡毒支原体阳性血清发生凝集;分子生物学鉴定结果显示,所扩增的核酸序列与鸡毒支原体匹配度高达99%;MIC测定结果显示,中药黄连和黄柏对分离的鸡毒支原体的抑菌活性较强,属敏感范畴,其MIC分别为≤0.98和0.39 mg/mL,而中药金荞麦和鱼腥草对鸡毒支原体中度敏感,MIC均≥125 mg/mL,板蓝根、白鲜皮、当归、艾叶对鸡毒支原体均不敏感。综上所述,本研究成功分离到1株鸡毒支原体,并且筛选出4种对该菌株敏感的中药,分别为黄连、黄柏、金荞麦和鱼腥草,为后期进一步研究防治鸡毒支原体病的中药组方时提供参考依据。     

不同地区鸡毒支原体的分离鉴定及药物敏感性研究

【目的】了解中国不同地区鸡毒支原体（Mycoplasma gallisepticum，MG）的流行和耐药情况，为禽支原体病的监测与防控提供科学依据。【方法】在广东、福建和安徽3个省份疑似MG感染的养殖场采集的43份气囊样品中分离培养和纯化MG流行菌株，并对其进行培养特性观察、生化及染色鉴定、血清学特异性鉴定、PCR测序分析、致病性试验和对常见抗菌药物敏感性试验。【结果】分离到3株疑似MG，均可使培养基颜色变黄，在固体培养基上呈典型"煎蛋"样菌落，可发酵葡萄糖，不水解精氨酸，不能利用尿素，符合MG培养特性。姬姆萨染色观察菌体形态、血清学特异性鉴定结果进一步证实分离株为MG。mgc2基因测序结果显示，3株分离株与强毒代表株亲缘关系更近。致病性试验结果显示，3株分离株与MG感染临床症状一致，发病率为70%~90%，致病力较强。药物敏感性试验结果显示，3株分离株均对恩诺沙星、替米考星、土霉素、氟苯尼考耐药，对泰万菌素和沃尼妙林均敏感，对大观霉素、金霉素、泰乐菌素和泰妙菌素的敏感性具有地域差异：福建株对大观霉素表现明显耐药，安徽株对泰妙菌素表现耐药，而广东株对大观霉素、金霉素和泰妙菌素均较敏感。【结论】本试验成功分离到3株MG，致病力较强，且均存在一定程度耐药，对药物的敏感程度有地区性差异，今后仍需加强各地区MG的药物敏感性监测，减少耐药性的产生与扩散。     

鸡毒支原体不同地区分离株对常用抗菌药物的敏感性试验

从广东、四川和北京等地疑似鸡毒支原体感染鸡中分离并利用种特异性基因（fMG-2）PCR方法鉴定了51株鸡毒支原体,并测定了这些分离株对常用抗菌药物的敏感性。结果表明,三个地区临床分离鸡毒支原体对泰乐菌素仍然保持较好的敏感性,可作为鸡毒支原体感染的首选药物。三个地区的鸡毒支原体对林可霉素和庆大霉素敏感性显著下降,广东、四川两地分离株对强力霉素也有明显耐受性,部分广东分离株对氟喹诺酮类药物敏感性显著下降。     

耐氟喹诺酮类药物鸡毒支原体DNA回旋酶及拓扑异构酶Ⅳ的突变特征

采用二倍稀释法测定临床分离的4株鸡毒支原体对常用抗菌药物的敏感性,PCR方法和基因测序法对鸡毒支原体DNA回旋酶编码基因gyrA、gyrB及拓扑异构酶Ⅳ编码基因parC和parE耐药决定区进行分析。敏感性测定结果表明,4株分离鸡毒支原体对泰乐菌素、泰妙林、沃尼妙林和替米考星有很高的敏感性,对四环素和红霉素中度敏感,对林可霉素、氟苯尼考和氟喹诺酮类药物呈现不同程度的耐药性。4株耐氟喹诺酮类药物鸡毒支原体均在GyrA和ParC的喹诺酮类耐药决定区(QRDR)发生氨基酸的改变,GyrA的氨基酸取代模式有两种,分别为Ser81→Gly和Ser83→Ile,ParC仅在80位发生氨基酸取代(Ser80→Leu),GyrB和ParE均未发生氨基酸改变。     

几种动物病原菌对替米考星耐药性的体外诱导

在测定替米考星对鸡毒支原体、多杀巴氏杆菌和猪胸膜肺炎放线杆菌的最小抑菌浓度基础上,采用药物浓度递增法体外诱导3种病原菌对替米考星的耐药性。结果鸡毒支原体经9次传代对替米考星产生了高水平耐药,多杀巴氏杆菌经7~9次传代对替米考星产生了高水平耐药,猪胸膜肺炎放线杆菌经14次传代对替米考星的耐受浓度未发生明显变化;鸡毒支原体和多杀巴氏杆菌替米考星诱导耐药株对红霉素、阿奇霉素、泰乐菌素、吉他霉素和林可霉素表现交叉耐药。研究结果提示,替米考星较易诱导鸡毒支原体和多杀巴氏杆菌产生耐药性,兽医临床应合理应用。     

一株鸡毒支原体致病性研究与药敏试验

为进一步掌握鸡毒支原体的致病性,从某养殖场分离一种鸡毒支原体的毒株,开展致病性试验和药敏试验。选择使用20枚孵化5日龄的SPF鸡胚,使用2种不同浓度的菌液进行攻毒试验,观察鸡胚的死亡情况和解剖情况,并按照支原体培养方法分离得到病原后进行常规药敏试验,确定哪种抗生素对支原体敏感。研究结果表明,不同浓度均会造成鸡胚死亡,低浓度鸡胚死亡率70%左右,高浓度造成鸡胚全部死亡。分离得到的毒支原体对盐酸沃尼妙林、盐湖索酸泰妙菌素、强力霉素敏感性最强,其次是盐酸大观霉素、水溶性氟苯尼考、酒石酸泰乐菌素。研究结果可知,分离得到的一株鸡毒支原体的致病性较强,同时分离出的高敏药物对支原体具有很强的抑制作用。     

柔嫩艾美耳球虫地克珠利耐药株与敏感株基因组SNP杂合度的比较分析

鸡球虫病是阻碍养禽业健康发展的重要寄生虫病。地克珠利作为一种低毒、广谱、高效的抗球虫药物得到了广泛使用,但长期使用导致鸡球虫耐药株广泛出现。为了探究鸡球虫对地克珠利产生耐药性的分子机制,本试验采用药物浓度递增的方法诱导耐药株,通过全基因组重测序分析柔嫩艾美耳球虫耐药株和敏感株之间单核苷酸多态性(SNP)的杂合度差异。结果显示,与敏感株相比,耐药株在蛋白激酶(EVM0000192)基因存在2个非同义突变,推测其与柔嫩艾美耳球虫的地克珠利耐药性的产生有关,本试验为后续利用更多耐药株的SNP来发掘地克珠利耐药基因提供了科学依据。     

副猪嗜血杆菌四川株生物学特性及药物敏感性研究

为了解副猪嗜血杆菌四川分离株的生物学特性及耐药性情况,本研究采用微生物及分子生物学技术对副猪嗜血杆菌(Hps)四川分离株从生物学特性、16S rRNA基因PCR扩增和耐药性方面进行了研究.结果显示,Hps四川分离株具有一定的微厌氧性,能产生"卫星现象",分离株与其他地区分离株生化特性基本一致;所有分离株均扩增出821 bp的特异性16S rRNA基因片段.药物敏感性试验结果显示,分离株对头孢拉定、氨苄西林、头孢氨苄、阿莫西林、头孢克洛、氧氟沙星、头孢噻肟、呋喃妥因具有较高的敏感性,敏感率为75.61%~87.80%;对林可霉素、羧苄青霉素、恩诺沙星、阿米卡星、磺胺甲唑均表现出较高的耐药性,且多重耐药严重,5~8种耐药占比最多,为21.95%~30.49%,有7株可耐受多达13种药物.结果表明,Hps四川分离株存在严重的耐药性现象.     

牛精子体外获能前后超微结构的研究

应用透射电镜技术,对牛精子体外获能前后超微结构观察的结果表明,牛精子的超微结构相似于其他哺乳动物,但顶体较大;获能后精子发生了顶体反应.在顶体反应中,囊泡化有3种形式:(1)顶体外膜相互融合,形成囊泡;(2)顶体外膜内陷形成囊泡化;(3)顶体外膜和质膜融合形成囊泡.此外,对精子的结构特点和囊泡形成过程进行了讨论.     

Ultrastructural ocular lesions of 6-aminonicotinamide toxicosis in rabbits

6-Aminonicotinamide, given by intraperitoneal injection to male and female Dutch belted rabbits, produced swelling and vacuolation of ciliary and iridal epithelium plus vacuolation of the retinal pigment epithelial and outer plexiform layers of the retina. By transmission electron microscopy, inner and outer ciliary epithelial cells and inner iridal epithelial cells contained numerous coalescing, membrane-bound vacuoles of the cytocavitary network. These vacuoles were viewed as numerous interconnecting, intracytoplasmic cavities in scanning electron micrographs. Swelling of vacuolated epithelial cells and the presence of fibrin and proteinaceous fluid in the ciliary stroma resulted in thickening of the anterior ciliary processes with the formation of surface alterations detectable by scanning electron microscopy. In transmission electron micrographs the vacuoles in the retinal pigment epithelium were large, electron-lucent spaces and the vacuoles in the outer plexiform layer of the retina appeared to be intracytoplasmic spaces in axons of photoreceptor cells. Distention of cytocavitary structures has been reported in glial cells of animals given 6-aminonicotinamide and this change was apparently due to alterations in ion and water movement across cellular membranes that resulted in intracellular edema.     

Inner structures of some coronaviruses.

When treated with formaldehyde, Tween 80, sodium oleate and Nonidet P-40, avian infectious bronchitis virus, porcine transmissible gastroenteritis virus, neonatal calf diarrhea coronavirus, porcine hemagglutinating encephalomyelitis virus as well as the human coronavirus show similar inner structures by negative staining. The first one is an inner membranous bag. This structure could be evaginated following treatments used and does not show the characteristic projections of coronaviruses. Subsequently, the inner fold could be separated from the outer membrane at the point of junction between these two membranes. Each virus does not react in the same way to the action of the different products. The transmissible gastroenteritis virus appears more sensitive to treatments than other viruses. On the other hand, the hemagglutinating encephalomyelitis virus is the most resistant. The variable sensitivities of these viruses are not related to the type of host-cells. Also, a second internal structure, which is more dense than the viral particle, encircles partially the aperture of the internal tongue-shaped structure and seems to emerge from the viral particle through the aperture of the inner bag.     

鸡柔嫩艾美耳球虫不同抗药性虫株的种内多态性研究

应用RAPD技术进行柔嫩艾美耳球虫4个单一抗药性虫株与1个敏感株的基因组DNA多态性分析，发现4个抗药性虫株及敏感株之间的相似值均大于99%；OPAO3引物对抗盐霉素株扩增出一条约500bp的特异条带，OPH02引物对抗克球粉株和抗盐霉素株均扩增出了约1kb的第三条主带，这些特异条带的出现很可能与球虫抗药性基因有关，有可能用于抗药性虫株的诊断与鉴定。     

马杜霉素对不同虫株柔嫩艾美耳球虫超微结构的影响

鸡体内用5mg/kg马杜霉素作用于柔嫩艾美耳球虫（Eimeria tenella)敏感虫株和抗药虫株，比较裂殖生殖阶段超微结构的差异。敏感株型裂殖体体积变小，所生成裂殖子的数目变小，裂殖子膨胀，裂殖子间（带虫空泡内）微管膨胀，结构模糊，有髓鞘样结构出现；限制膜外突和细胞质分离形成空隙，膜结构模糊，有破损；细胞浆空泡化，出现附加体和髓鞘样结构；少数细胞核的外膜外突和内膜分离，有的部位的核膜模糊、破损。首次发现裂殖子顶体结构发生异常变化，棒状体消失，微线数目减少或者消失；线粒体表现为形态改变，嵴脱落，内部形成空泡、膜状结构和髓鞘样结构，线粒体的嵴与长辆平行排列，且贯穿线粒体。抗药虫株没有发现异常变化。推理抗生素类抗球虫药的作用机理为破坏虫体结构和抑制虫体的生理机能两个方面；球虫对该类药物产生抗药性的机制是基因突变和药物选择的结果。     

Morphology of the Dentin Structure of Sloths Bradypus tridactylus: A Light and Scanning Electron Microscopy Investigation

The aim of this study was to describe the dentine morphology of sloths (Bradypus tridactylus). The sloth teeth were removed and prepared for light microscopy (LM) and scanning electron microscopy analyses (SEM). LM revealed two patterns of tubular dentins: an outer with dentinary tubules over the all tooth length and one in the inner part with larger diameter and more spaced tubules, when compared to those present in the outer dentine. These findings were confirmed by SEM, which revealed a tubular pattern in the outer dentine like in humans. The inner dentine displayed pared grouped tubules that were characterized as vascular channels. It can be concluded that this sloth species present two types of dentins: an inner dentin (ortodentin) and an outer dentin characterized as a vascular dentin. This suggests a partial evolutive/adaptive process of this dental tissue, as compared to other mammalian species.     

基于植物营养的荔枝龙眼果实异常现象的初步诊断

近年我国荔枝龙眼主产区出现多起荔枝龙眼果实发育期果实异常现象。小果和中果期表现为外果皮出现黑褐色斑点,内果皮黑褐色斑块更为明显,严重者果皮开裂,种胚坏死。大果期外果皮皱缩凸起,果实畸形变小,内果皮有黑褐色或淡绿色向果肉侵入的痂状物,严重者侵入部位果肉褐变腐烂。不同时期果实异常症状均为内部重于外部。在排除病原物侵染致病前提下,观察对比了异常果和正常果果实微观形貌,测试了果实不同部位N、P、K、Ca、Mg和B等养分含量,并调查了果园的管理、施肥及气候情况。初步判断这些果实异常现象是由不良气候引起的果实缺Ca生理性病害。对个别果园,缺B可能也是一个共同影响因素。     

Prenatal development of retina in buffalo (Bubalus bubalis)

The development of retina in Indian buffalo (Bubalus bubalis) has not been reported previously. The aim of the present study was therefore to report the major landmarks and the time course in the development of retina. Serial histological sections of Indian buffalo embryos and foetuses were used as group1 (<20.0 cm CVRL), group2 (>20.0 but <40.0 cm CVRL) and group3 (>40.0 cm CVRL). Age estimation was made on the basis of crown vertebral‐rump length (CVRL), which ranged between 36 and 286 days (1.6–94.0 cm). The retina in Indian buffalo was developed in a similar manner to that of the other mammals with the principal differences in the time of occurrence of various layers of this nervous tunic. In 36 days (1.6 cm stage), the foetal retina was composed of pigmented layer and the layer of neuroblasts. Differentiation of layers was first observed in 47 days (4.0 cm CVRL) which became prominent in 52 days (5.1 cm stage). At 120 days (20.5 cm stage), the differentiation of inner plexiform layer and inner nuclear layer was evident. At 143 days (31.0 cm) foetal age, the faint line in neuroblastic layer was the first evidence of the future outer plexiform layer. In foetuses of group III, the retina was comprised of all 10 layers (eight cell layers and two membranes) viz. pigmented epithelium, layer of rods and cones, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, layer of nerve fibres and the inner limiting membrane.     

Organic matrix morphology and distribution in the palisade layer of eggshells sampled at selected periods during lay

1. 1 cm2 pieces of eggshells from a commercial battery flock were plasma etched to remove the outer shell membranes. 2. They were decalcified using EDTA (200 g/l, pH 6.9 to 7.0) in paraformaldehyde (20 g/l) and 25% gluteraldehyde (20 ml in 0.98 l) in phosphate buffer, then prepared for light and transmission electron microscopy. 3. Light microscopy revealed a differential distribution of matrix material within all 3 regions of the palisade layer at the beginning of lay. 4. Transmission electron microscopy revealed a more even distribution of matrix at the beginning of lay, although morphological differences were observed. At the end of lay all 3 regions showed an increase in % matrix and vesicles/10 cm2 of micrograph compared to the middle and beginning of lay periods. 5. It is hypothesised that matrix vesicles are involved in the regulation of the physiochemical environment within the forming eggshell and that the decline in shell quality associated with the end of lay is related to a concomitant change in matrix quality.     

Development of the Retina in the Porcine Fetus A Light Microscopic Study

Morphogenesis of the porcine retina was studied using light microscopy from 4 weeks of gestation until birth (18 to 310 mm crown-rump length), and compared with the adult stage (6 months). Tissue samples were examined from the posterior and peripheral parts of the retina. At 18 mm the retina consists of an inner marginal layer and an outer layer of neuroblastic cells. At 18-40 mm the latter layer is divided into an inner and an outer neuroblastic layer by the transient layer of Chievitz. Subsequently, the development of the different retinal layers begins at the inner retinal border and moves progressively outwards; it also spreads from the posterior to the peripheral part of the neural retina. Many cells of the inner neuroblastic layer are prospective ganglionic cells which migrate inwards, thus forming the ganglion cell layer and the inner plexiform layer at 90 mm. At 120 mm, primitive horizontal cells appear within the outer neuroblastic layer. Separation of this layer into the inner nuclear, outer plexiform and outer nuclear layers is first evident at 180 mm. At this stage all retinal layers are present, except the layer of the photoreceptor cells which is not widespread until at 220 mm. The inner and outer segments of the photoreceptor cells lengthen considerably during the last month of gestation. During the late fetal stage the nerve fiber layer, the inner and outer plexiform layers and the layer of rods and cones all continue to increase in thickness. Concurrently, the ganglion cell layer and the inner and outer nuclear layers have reached their maximal thickness and become thinner. After the total thickness of the neural retina amounts to approximately 180 microns at two to three weeks before birth, it then thins to approximately 160 microns in the adult stage.