广东规模化鸡场死鸡胚中鸡毒支原体的分离鉴定、致病性及药物敏感性

旨在从广东某规模化鸡场死鸡胚进行鸡毒支原体(Mycoplasma gallisepticum,MG)的分离,并进行遗传进化分析、致病性及药物敏感性研究。本研究从广东某规模化鸡场的带菌死胚卵黄组织中分离禽支原体,通过菌落观察、血清学试验、16S rRNA支原体通用引物序列鉴定等方法,对分离的菌株进行种属鉴定,同时将分离的毒株对鸡胚和SPF鸡进行攻毒试验及抗菌药物敏感性试验。结果显示：显微镜下菌落呈典型的“荷包蛋”状。平板凝集试验显示其与MG阳性血清有凝集反应,与MS阳性血清不反应;16S rRNA序列测序发现各分离株与MG相似性达99.9%,因此确定分离株为MG。将各分离株感染7日龄SPF鸡胚,结果显示感染鸡胚大多于临近出壳时死亡;感染3周龄的SPF鸡,3周后解剖发现鸡气囊发生显著病理变化,说明其具有较强致病力;药物敏感性试验结果显示,5株分离株对盐酸沃尼妙林、多西环素、泰万菌素及泰妙菌素有较高敏感性,对替米考星、泰乐菌素、恩诺沙星、红霉素、吉他霉素、林可霉素呈现出不同程度耐药性。综上,从广东某规模化鸡场死鸡胚中成功分离到5株MG分离株,各分离株均可引起红细胞凝集,导致鸡胚死亡,对SPF...     

鸡滑液囊支原体的流行调查及不同地区分离株药物敏感性分析

为了解近年来鸡滑液囊支原体感染的流行情况,自2015年1月-2017年12月,对广东、广西、江苏、浙江、福建等地10个不同地区养殖场的感染情况进行调查分析。结果显示,该病在调查的10个不同地区均有发生,感染鸡群多见于l～4月龄,平均感染日龄为60日龄;8个不同品种鸡的平均感染率为8.89%,平均死亡率为2.16%。感染率和死亡率均存在品种差异,其中817肉杂鸡及清远麻鸡的感染死亡率最高。从157份疑似鸡滑液囊支原体感染鸡的病料中分离到96株田间流行株,各地区随机取1株进行10种常用抗菌药物的敏感性测定。结果表明,10株临床分离株均对泰妙菌素、泰万菌素、泰乐菌素及多西环素较为敏感,对金霉素、庆大霉素等的敏感性显著下降且出现不同程度的耐受性,对盐酸环丙沙星、沙拉沙星敏感性较差,此结果可为临床合理用药提供参考。     

鸡滑液囊支原体不同地区分离株对常用抗菌药物的敏感性试验

为调查不同地区鸡滑液囊支原体的耐药情况,从河南、江苏、安徽、河北和广东五省204份疑似鸡滑液囊支原体感染鸡的病料中分离到20株鸡滑液囊支原体,每省随机取1株进行了12种常用抗菌药物的敏感性测定。结果表明,5株鸡滑液囊支原体均对泰乐菌素较为敏感,而对恩诺沙星、氧氟沙星、盐酸环丙沙星具有不同程度的耐受性。本实验可为临床合理用药提供参考。     

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。     

潍坊地区鸡毒支原体的流行病学调查

为了解鸡群中鸡毒支原体的流行现状,对该病防控措施的制定及药物筛选提供科学依据,从潍坊地区76所肉鸡场的疑似病料进行鸡毒支原体分离。采用瑞氏-姬姆萨染色及PCR鉴定等方法,共鉴定出鸡毒支原体33株,分离阳性的病例多数在临床中都出现了明显的呼吸道症状,剖检时可见不同程度的大肠杆菌的混合感染,对支原体分离株进行测序,并进行了序列分析。结果表明,抑制杂菌的生长是鸡毒支原体分离成功的关键因素,流行病学数据证实鸡毒支原体感染目前在养鸡场中仍然非常普遍,混合感染的发生导致鸡群死亡率上升。     

山东地区发生呼吸道性疾病的肉鸡场鸡毒支原体的分离鉴定

为了解山东省发生呼吸道疾病的肉鸡场鸡毒支原体的感染情况,2013年至2014年从山东地区发生呼吸道疾病的商品肉鸡场采集发病鸡,记录其临床表现(发病特点、临床症状和病理变化),并进行鸡毒支原体分离鉴定。对符合鸡毒支原体培养特性的分离株通过PCR扩增和测序加以鉴定,共分离鉴定出36株鸡毒支原体。3~20日龄肉鸡采集23场,分离出3株鸡毒支原体,分离阳性的样品疑来自鸡毒支原体单纯感染的病例;21~40日龄肉鸡采集78场,分离出33株鸡毒支原体,分离阳性的样品均来自混合感染病例。     

鸡毒支原体的分离与鉴定

从广东、四川及北京地区患有慢性呼吸道疾病的不同鸡场采集194份样本,接种FM-4培养基进行分离培养。对符合鸡毒支原体培养特性培养物采用PCR加以鉴定,结果培养鉴定出69株鸡毒支原体,其分离率为35.57%(69株/194株)。     

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。     

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。     

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。     

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship.     

Identification of Mycoplasma gallisepticum by use of monoclonal antibody in a rapid slide agglutination test.

Monoclonal antibody (MAb) against Mycoplasma gallisepticum strain PG31 was produced in BALB/c mice. The MAb (designated M9) was of IgG3 isotype and reacted with an epitope in M gallisepticum antigens with molecular weights of 35, 90, 95, and 98 kilodaltons (kDa). The M9 reacted with M gallisepticum antigens in the dot-blot ELISA and in western blot assays. It agglutinated M gallisepticum strains PG31, F, R, S6, A5969, and 9 field isolates from various sources. A coagglutination assay, using Staphylococcus aureus (Cowan strain 1), was developed to enhance the agglutination of some weakly agglutinating M gallisepticum isolates. The M9 did not react with M synoviae, M iowae, M meleagridis, M gallinarum, or M gallinaceum in any of the aforementioned assays. This MAb may be useful in facilitating laboratory diagnosis of M gallisepticum infections.     

In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys

Monitoring of susceptibility to antibiotics in field isolates of pathogenic avian mycoplasmas is important for appropriate choice of treatment. Our study compared in vitro susceptibility to enrofloxacin and difloxacin in recent (2005-2006) isolates of Mycoplasma gallisepticum and Mycoplasma synoviae from meat-type turkey flocks with archived (1997-2003) isolates and reference strains. Comparison of minimal inhibitory concentration (MIC) values determined by microtest, agar dilution and commercial Etest showed good agreement, but underscored the need for standardized methods for testing. Notably, while the commercial Etest was convenient and accurate for determining MICs for enrofloxacin in the range 0.002-0.094mug/ml, the endpoint of inhibition for M. gallisepticum and M. synoviae strains with MIC values >/=1.0mug/ml could not be determined. A decrease in susceptibility to both fluoroquinolones was detected in archived strains but to a greater degree in recent isolates, most of which had MICs above the NCCLS susceptibility breakpoint for these antibiotics (相似文献   

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease with almost 20 per cent mortality in pheasants, whereas the infection was less pathogenic for chickens and turkeys reared at the same site. The M gallisepticum isolates from pheasants had a unique pMGA gene sequence containing a repeat of 12 nucleotides, and they contained only small amounts of the cytadhesins MGC1 and MGC3 and no PvpA protein. However, they expressed some typical M gallisepticum proteins and several proteins which were immunogenic for pheasants, chickens and turkeys. A strain of M gallisepticum isolated from the sinus of a pheasant was highly pathogenic for chicken embryos. In the second case, the M gallisepticum strain that was associated with respiratory disease and mortality in peafowl also affected chickens. M gallisepticum strain ULB 992 was isolated from the infraorbital sinus of a dead peafowl. The ULB 992 strain synthesised a small amount of MGC3, a truncated form of MGC1 and lacked PvpA. However, it expressed several proteins which were immunogenic for the birds infected with M gallisepticum at both gamebird operations.     

Molecular characterization of Mycoplasma gallisepticum isolates from turkeys

Mycoplasma gallisepticum was isolated from several turkey flocks at different locations in the United States that were clinically affected with respiratory disease. Five of these isolates from four series of outbreaks had patterns similar to the 6/85 vaccine strain of M. gallisepticum by random amplified polymorphic DNA (RAPD) analysis using three different primer sets, whereas with a fourth primer set (OPA13 and OPA14), only two of the isolates were similar to 6/85. Results obtained by sequencing portions of the pvpA, gapA, and mgc2 genes and an uncharacterized surface lipoprotein gene indicated that the field isolates had DNA sequences that ranged from 97.6% to 100%, similar to the 6/85 results. In some of the outbreaks there was an indirect association with the presence of commercial layers in the area that had been vaccinated with this vaccine strain, but there was no known close association with vaccinated birds in any of the outbreaks. Turkeys were challenged with two of the field isolates and with 6/85 vaccine strain. Turkeys challenged with the field isolates developed respiratory disease with airsacculitis and a typical M. gallisepticum antibody response, whereas birds challenged with 6/85 developed no respiratory signs or lesions and developed only a weak antibody response. Although these isolates were very similar to the 6/85 vaccine strain, it was not possible to prove that they originated from the vaccine strain-it is possible that they could be naturally occurring field isolates.     

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.     

Antigenic variation of Mycoplasma gallisepticum, as detected by use of monoclonal antibodies.

A panel of monoclonal antibodies (MAb) developed against Mycoplasma gallisepticum strain PG31 was used to probe the antigenic profiles of 5 recognized strains (PG31, R, S6, F, A5969) and 6 field isolates of M gallisepticum. Monoclonal antibody G9 predominantly recognized antigens at apparent molecular mass positions of 90 to 98 kDA. The MAb reacted with all strains and isolates, but the molecular mass position of the antigens varied among some mycoplasmas. Monoclonal antibody G12 reacted with all strains and isolates of M gallisepticum and had an identical banding pattern. However, MAb G10 and G11 reacted selectively only with a limited number of strains and/or isolates. Surface distribution of the MAb-recognized antigens was revealed by immunoelectron microscopy. Partial physicochemical characterization of MAb G9-recognized antigens identified glycopeptide characteristics. Monoclonal antibody G9 reacted with surface antigens and, hence, participated in agglutination of M gallisepticum. However, the degree of agglutination varied among the various strains and isolates, indicating a quantitative or conformational limitation or an alteration in the anomeric expression of the epitopes. Antigenic variation in M gallisepticum may be mediated by immunologic selective pressures, or a proclivity for habit niche in the host.     

Complementary randomly amplified polymorphic DNA (RAPD) analysis patterns and primer sets to differentiate Mycoplasma gallisepticum strains.

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate 7 strains of Mycoplasma gallisepticum. Six commercially available primers or primer combinations were screened for their ability to differentiate vaccine and type strains. Although major and minor bands were produced with each primer, many of the primers were unsuitable for strain differentiation. The use of primer 6 and combined primers 3 and 4 resulted in complementary RAPD banding patterns for each M. gallisepticum strain. Eleven different isolates representing 7 different strains were segregated into 7 different patterns, corresponding to the 7 strains.     

柔嫩艾美耳球虫野外抗药性虫株的RAPD分析

用RAPD方法对柔嫩艾美耳球虫（Eimeria tenella）的8个分别来源于黑龙江、北京、四川、甘肃和广东的中山、新会、东莞以及广州近郊的江村镇的野外分离虫株和2个药物敏感性虫株进行了遗传多态性分析。同时用9种抗球虫药物即马杜霉素（5mg/kg）、盐霉素（60mg/kg）、莫能霉素（120mg/kg）、拉沙菌素（90mg/kg）、克球多（150mg/kg）、常山酮（3mg/kg）、氯氢苯乙氰（1mg/kg）、尼卡巴嗪（125mg/kg）和球痢灵（125mg/kg）分别对8个野不进行抗药性试验，8个虫株对上述药物均有不同程度的抗药性。RAPD分析表明：10个样品都有相似而清晰的主带，每个泳道有5－13条带不等，大小为0.18-2.10kb。它们之间的相似率（SI）大于40.58%，最大的为90.72%，这种相似率属种内变异水平。根据SI值可把10个样品划分为3个类群：广东虫株类群，群内比较，平均SI值为76.60%；广东省外虫株类群，群内比较的平均SI值72.14%；敏感株类群，其间的SI值为89.27%。在另一方面，广东株与广东省外株、敏感株之间比较，其SI的平均值分别仅为63.03%和47.25%，说明柔嫩艾美耳球虫抗药性虫株之间有遗传差异法。