鸡毒支原体的分离培养和初步鉴定

从济南地区某疑似慢性呼吸道病(CRD)的鸡场分离到1株支原体菌株,经菌落形态观察、生化试验、血清学鉴定,证明该分离株为鸡毒支原体(MG)。     

鸡毒霉形体pMGA基因中TGA的同义突变及其原核表达

为了进一步研究鸡毒霉形体黏附素蛋白(pMGA)基因的生物学特性,利用PCR对该基因进行分段扩增,同时同义突变编码色氨酸的TGA及另外2个氨基酸的编码子以便形成酶切位点.将扩增片段连入pMD18-T载体获得含不同长度片段的质粒pMD-Ⅰ,pMD-Ⅱ,pMD-Ⅲ和pMD-Ⅳ,利用限制性内切酶消化pMD-Ⅰ和pMD-Ⅱ及pMD-Ⅲ和pMD-Ⅳ后回收相应的片段并进行连接获得pMD-Ⅴ(Ⅰ Ⅱ)和pMD-Ⅵ(Ⅲ Ⅳ).再用限制性内切酶消化pMD-Ⅴ和pMD-Ⅵ,回收Ⅴ和Ⅵ,并将其插入表达载体pGEX-KG中构建了含突变基因的GST融合表达载体.同时分别构建了片段Ⅲ及Ⅵ的GST融合表达载体.在IPTG诱导下,构建的表达载体在BL21(DE3)中获得了高效表达.表达产物相对分子质量大小分别为92000、39000、60000,经Western blotting分析证明均具有免疫原性.     

An ultrastructural study on the interaction of Mycoplasma gallisepticum with the chicken tracheal epithelium

Seven-day-old chickens wee intratracheally inoculated with Mycoplasma gallisepticum. The tracheas collected 6 and 14 days after chickens were inoculated were subjected to titration of mycoplasma and examination by light and electron microscopy. The mycoplasma organisms grew well; 10(7) to 10(8) color-changing units in a milligram of tissue were determined. Tracheal lesions occurred in close association with the presence of mycoplasmas and were characterized by degeneration of the epithelial cells and inflammatory cellular infiltration of the mucosa. Mycoplasmas were predominantly found extracellularly and only rarely in phagocytic vacuoles of the epithelial cells. Although the mycoplasmas exhibited considerable pleomorphism in size and shape, most of them were oval or round, and the largest diameters were between 300 and 700 mn. Elongated and irregular forms were also observed, particularly in those mycoplasmas adhering to the epithelial cells. The organism had a limiting unit membrane, the fibrillar nuclear area, the peripheral cytoplasmic area containing numerous ribosomes, and a terminal bleb structure. Mycoplasmas attached to the epithelial cells by their blebs close to the host cell membrane. At the attachment site, neither fusion of the membranes of the mycoplasma and host cell nor injury to the host cell membrane could be demonstrated. Nevertheless, seemingly, the intimate association between the adhering mycoplasmas and the epithelial cells might be an important factor in pathogenesis of the disease.     

鸡毒霉形体HS株pMGA多基因族的研究

鸡毒霉形体（Mycoplasma gallisekpticum,GM)基因组中存在着编码细胞表面血凝粘附素蛋白（protein of Mycoplasma gallisepticum adhesin,pMGA)相关基因组的多基因族。为筛选出含有完整的pMGA基因片段，并估算出鸡毒霉形体HS株基因组中pMGA多基因族的基因数，本试验以pUC18为载体，用EcoR I限制性内切酶构建了鸡毒霉形体HS株的基因组文库。根据pMGA基因引导序列的保守区和pMGA基因间隔区的序列，人工合成了2个寡核苷酸探针（探针I、探针Ⅱ），经标记后，双筛选所建的基因文库，从600个转化子中共得到的38个含有pMGA基因的阳性克隆子，选其中1个7．5kb的片段（17号阳性克隆子，W17），用4种限制性内切酶（EcoR I,Hind Ⅲ，Pst I,Bgl Ⅱ）酶切消化，绘制了该片段的物理图谱。该片段酶切产物经电泳、转膜后与探针I和探针Ⅱ杂交，发现2个探针杂交图谱基本相同，根据杂交带及放射信号的强度值，估测出该片段含有4个pMGA基因，以这个片段所含的pMGA基因数为标准，估算出鸡毒霉形体HS株含有39个pMGA基因。     

Chick embryo tracheal organ: a new and effective in vitro culture model for Cryptosporidium baileyi

The economic impact of anthelmintic resistance was investigated in lambs by comparing productivity parameters in groups of animals treated either with a highly effective anthelmintic, or an anthelmintic to which three species of resistant worms were known to be present. Ten farmlets, each stocked with 30 lambs, were rotationally grazed for 5 months, with monthly treatments of either albendazole, to which resistance existed, or a new combination product containing derquantel and abamectin (DQL-ABA) to which there was no resistance. Stock on five farmlets were treated with each anthelmintic and productivity measures, including liveweights, body condition and faecal soiling were assessed throughout. In addition, fleece weights and information on carcass weight and quality was collected at the end of the trial. Anthelmintic efficacy was measured at the last two treatment dates by faecal egg count reduction test with larval cultures. Albendazole demonstrated efficacies of 48.4% and 40.9% for Trichostrongylus spp. and Teladorsagia circumcincta respectively. By contrast, the DQL-ABA treatments were >99% effective against all genera. The difference in live-weight gain was 9 kg in favour of the DQL-ABA treatments. This translated into a 4.7 kg increase in carcass weight with a 10.4% increase in carcass value. Significant differences in body condition scores, faecal breech soiling and fleece weights were also recorded, all in favour of the DQL-ABA treatments. The time required for 50% of the animals to reach a target live-weight of 38 kg was significantly shorter (by 17 days) in those animals treated with DQL-ABA. The results show that the production cost of subclinical parasitism as a result of using an anthelmintic product which is less than fully effective due to resistance can greatly exceed the cost of routine testing of anthelmintic efficacy and the adoption of new anthelmintic classes. There is a strong case for many farmers to re-evaluate their position on some of these issues in order to optimise financial performance.     

鸡毒霉形体HS株pMGA多基因族序列分析

用限制性核酸内切酶对鸡毒霉形体(MG)HS株基因文库中经初步估测可能含有的4个鸡毒霉形体粘附蛋白(protein of Mycoplasma gallisepticum adhesin,pMGA)基因的MgW17重组质粒进行了不同组合的酶消化,根据消化片段的大小及酶切位点绘制了7.5kb外源片段的物理图谱;然后根据所得的物理图谱,选用Pst I和EcoR I将MgW17外源片段酶解成1.3、2.0、4.2 kb 3个部分,将它们分别亚克隆到SK(十)质粒上,得到了3个亚克隆子SGp100、SGp200、SGp300.使用核酸外切酶Ⅲ缺失法构建缺失子系列,经序列分析后得到MgW17外源片段的全部序列.序列全长7 434 bp,中间含有2个完整的pMGA基因和另2个不完整的pMGA基因的首部和尾部,分别将它们顺次命名为H-pMGA1.1、H-pMGA1.2、H-pMGA1.3和H pMGA1.4.2个完整的pMGA基因的阅读框长度分别为1 967 bp(1.2)和2039 bp(1.3);H-pMGA1.1首部不完整的阅读框的长度为720 bp,尾部不完整的H-pMGA1.4的长度为1 752 bp.将H-pMGA基因与已报道的MG.S6株、F株的pMGA基因进行了比较,探讨了pMGA基因在不同MG菌株中的相似性、基因启动子结构的差异、间隔区内GAA重复序列对转录调控的影响等.     

广东地区鸡毒支原体血清学调查

应用血清快速平板凝集方法,对广东地区12个非鸡毒支原体免疫的种鸡场及3个鸡毒支原体免疫的种鸡场进行了鸡毒支原体感染的血清学调查。结果显示:不同种鸡场及不同品种的种鸡的MG抗体阳性率高峰期均在11周龄左右,即鸡群的MG感染高峰期均在6周龄左右。     

Evaluation of cytopathologic changes induced in chicken tracheal epithelium by Mycoplasma gallisepticum in vivo and in vitro

Changes in tracheal epithelial surfaces induced by Mycoplasma infection in vivo and in vitro included release of mucous granules followed by exfoliation of ciliated and nonciliated epithelial cells. Light microscopy, scanning electron microscopy, and transmission electron microscopy confirmed that the loss of cilia from individual cells was infrequent. Epithelial cells typically lost their intercellular connections, rounded up, exfoliated, and then lysed--giving rise to a population of cellular organelles, such as mitochondria and cilia intermixed with mucus to form the exudate found within the tracheal lumen. Repair of the epithelial surface was effected by basilar epithelial cells differentiating and filling in the spaces formed by exfoliated cells. These cells were hypertrophied, nonciliated at 14 days after infection in vivo, and covered with microvilli. In sectioned material obtained during the infection, there was increasing epithelial thickness due to cellular infiltration and edema. Tracheal rings in vitro showed similar changes to those seen in vivo, except that exfoliation was more severe and occurred earlier. In addition, there were no cellular infiltration due to the lack of a vascular supply and only a small amount of mucus due to the smaller number of mucous cells available to release into the tracheal lumen.     

Efficacy of experimental inactivated mycoplasma gallisepticum oil-emulsion bacterin in egg-layer chickens

Six groups of white leghorn pullets were studied to determine the ability of beta-propiolactone-inactivated Mycoplasma gallisepticum (MG) oil-emulsion bacterins to counteract reductions in egg production caused by MG infection. The pullets were inoculated with 0.5 ml of MG bacterin subcutaneously in the neck at about 20 weeks of age and were challenged with MG near 28 weeks of age, when they were in peak egg production. Various challenge schemes with infectious bronchitis virus were used at the time of MG challenge to increase the reduction in egg production. MG bacterins afforded protection against moderate drops in egg production in at least three of the studies, where the unvaccinated challenged control hens exhibited reduced egg production.     

复方中药制剂"百呼清"对鸡毒支原体感染的药效学研究

该试验旨在观察新研制的中药制剂百呼清药效。试验将人工感染鸡毒支原体的15日龄肉鸡，按常规方法随机分为6组，每组每天给2次，连续5d，停药后继续观察10d。结果给组增重显著或极显著高于感染对照组（P＜0.05 或 P＜0.01），但与健康组比较差异不显著（P＞0.05）;给药组均能明显降低抗体阳性鸡毒支原体感染疗效显著，其作用优于或相当于泰乐菌素。     

Pathogenic effects on domestic poultry of a mycoplasma gallisepticum strain isolated from a wild house finch

Mycoplasma gallisepticum (MG) has been isolated from wild house finches. The pathogenic effects of MG finch strain (K4058) and MG R-strain were compared after exposure of chickens and turkeys. Gross and histologic lesions, reisolation of the organism, serology, and clinical disease were evaluated. Milder histologic and gross lesions, in addition to lower serologic titers, occurred in birds inoculated with the finch strain. Mortality, concurrent with clinical and gross respiratory signs and lesions, was observed only in chickens challenged with R-strain. Both the MG finch strain and MG R-strain were recovered from the respective challenge groups at 14 and 28 days postexposure. The results show that MG isolated from wild house finches may infect domestic poultry species but causes only mild disease and is less virulent than MG R-strain. Commercial enzyme-linked immunosorbent assay kits best detected the serologic response of chickens and turkeys to the MG finch strain.     

鸡毒支原体灭活油乳剂苗对蛋鸡的田间免疫试验

应用鸡毒支原体灭活油乳剂苗对蛋鸡进行田间免疫试验,证实该苗安全性和效果良好。免疫后在６个月内能有效抵御支原体野毒的感染。试验组与对照组相比,总发病率低２９．４％,产蛋率高１３．６％,产蛋高峰期延长２—４周,而且药物预防和治疗费用明显降低。     

Characterization of mycoplasma gallisepticum infection in captive house finches (Carpodacus mexicanus) in 1998

Since 1995, the epidemic of mycoplasmal conjunctivitis in eastern house finches has affected the Auburn, AL, house finch population. To better characterize the current status of this host-parasite interaction, we established a captive flock of 38 seronegative, healthy finches in fall 1998. After a minimum quarantine period of 4 wk, two Mycoplasma gallisepticum (MG)-infected house finches were introduced into this flock. Over a 12-wk period, the flock was captured every 2 wk and each bird was observed for conjunctivitis. Blood and choanal swabs were collected from each bird for serologic analysis and for the detection of MG by polymerase chain reaction. The infection spread rapidly through the flock just as it had in a similar study performed in 1996 at the height of the epidemic. Unlike the earlier study in which birds remained chronically infected, most of the birds in our study recovered rapidly, and only three of the birds died during the study. Two patterns of host response to infection with MG were observed. Twenty-seven birds (73%) experienced an acute conjunctivitis that resolved, and the birds appeared to clear the infection. Ten birds (27%) suffered prolonged clinical disease, and MG could be detected in these birds intermittently throughout the experiment. These results, in conjunction with our surveys of MG in the wild population, suggest an evolving host-parasite interaction.     

Interaction of Tritrichomonas foetus and the bovine oviduct in an organ culture model

Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. The mechanism by which T. foetus causes abortion in cattle is largely unknown. There are no studies of infection in the cow oviducts, almost all published papers are related to vagina infection and few articles focusing on the uterus. The aim of the present study was to establish a working model of bovine oviduct epithelial cells and submit these cells to Tritrichomonas foetus interaction. Twenty bovine oviducts were obtained from cows at a commercial abattoir and T. foetus was injected through the isthmus into the oviduct lumen. The whole oviduct was analyzed by scanning and transmission electron microscopy. The results reported here demonstrate that: (1) fresh whole oviducts can be used as a good model to study parasite-host cell interaction; (2) cow oviduct epithelium has been shown to consist of two cell types: ciliated and nonciliated secretory cells, and T. foetus displayed great specificity for the nonciliated cells localized in the deeper oviduct folds; (3) T. foetus adheres as single separate cells, and maintains the flagella externalized; (4) differently from T. vaginalis, T. foetus does not change its shape during the adhesion process; and (5) oviduct cells exhibited morphological characteristics of apoptosis after trichomonadal interaction.     

Comparative sensitivities of oviduct and tracheal organ cultures and chicken embryo kidney cell cultures to infectious bronchitis virus

In vitro studies with organ (oviduct and trachea) and chicken embryo kidney cell cultures were attempted to assess the pathogenicity of locally isolated infectious bronchitis virus (IBV-P:120) initially isolated from the oviduct of young chicks. In oviduct cultures infected with IBV, ciliary movements decreased as early as 24 hours postinoculation (PI), and on the 6th day ciliary movements ceased completely. Cytopathic changes were also noticed. Immunofluorescent antigen was detected from 1 to 6 days PI, the maximum being on the 3rd day. The characteristic microscopic changes in the oviduct explants were reduced by 24 hours PI and had completely ceased on the 5th day. Cytopathic effect and immunofluorescent antigen were present from 1 to 8 days PI, being maximum on the 5th day. Histological changes marked by loss of cilia, rounding of the epithelial cells, degeneration, and sloughing were detected from 2 to 8 days PI. Low-embryo-passaged (EP-7) IBV did not produce cytopathic effect on the chicken embryo kidney cell cultures. On the contrary, high-embryo-passaged (EP-14) virus produced cytopathic effect at the third tissue-culture-passage level.