一株猪肺炎支原体HN0613株的分离鉴定

采集疑似猪支原体肺炎病理变化的肺脏,经Friis液体、固体培养基培养、纯化,PCR、测序分析、生化鉴定、生长抑制、代谢抑制和致病性试验证实分离获得一株猪肺炎支原体菌株,命名为HN0613.该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养达108 CCU/mL;菌株有较强的毒力,可作为疫苗候选株进一步研究.该菌株的分离鉴定为研制猪支原体肺炎灭活疫苗奠定了基础.     

一株猪肺炎支原体的分离鉴定

从全国部分猪场采集到疑似猪支原体肺炎肺组织病料12份,提取DNA进行猪肺炎支原体PCR和多重PCR检测,将病料研磨后分离猪肺炎支原体,最终分离到1株疑似猪肺炎支原体;通过测序分析、形态观察、生化试验、血清学试验证实其为猪肺炎支原体。该菌株能适应人工培养基的培养,且传代生长良好,液体培养基中培养活菌滴度达109CCU/m L;菌株有一定的致病性,免疫原性好,可作为疫苗备用菌株,该菌株的分离鉴定为研制猪支原体肺炎疫苗奠定了基础。     

猪肺炎支原体DJ-166株的分离鉴定

从山西某猪场采集疑似猪支原体肺炎肺组织病料,接种CH培养基培养,克隆纯化获得一株菌株。该菌株经PCR、培养特性、生化特性和血清学特性试验鉴定为猪肺炎支原体,命名为DJ-166株。该分离菌株在人工合成培养基上传8代稳定后,活菌计数达到108CCU/m L;菌液培养物气管注射猪后,致病性较弱;免疫原性试验证明该分离菌株具有良好的免疫原性,此结论为猪支原体肺炎疫苗的研究提供了必要条件。     

猪肺炎支原体强毒株的分离和鉴定

从15份疑似猪支原体肺炎病料中分离出病原,然后进行培养传代,再做染色镜检、PCR鉴定、序列分析和动物回归实验,最终将分离株接种于健康仔猪,结果仔猪出现典型的猪支原体肺炎临床症状和病理变化,证明该分离株为猪肺炎支原体.     

不同厂家猪血清配制的培养基培养猪肺炎支原体的比较研究

根据培养物的生长速度和含菌量方面筛选适合猪肺炎支原体CJ株生长的猪血清。配制改良Friis培养基时分别添加6种不同生产厂家的猪血清,由6种不同厂家猪血清配制的改良Friis培养基培养猪肺炎支原体CJ株。从生长时间可知,由厂家1的猪血清配制的改良Friis培养基更适宜猪肺炎支原体CJ株的生长。从测定的含菌量测定结果可知,由厂家1的猪血清配制的改良Friis培养基培养猪肺炎支原体CJ株的含菌量较高。结果表明,由厂家1的猪血清配制的改良Friis培养基培养猪肺炎支原体CJ株的生长时间短且含菌量高。     

猪肺炎支原体检测芯片的研制及初步应用

本研究探索建立一种新的基因芯片方法检测和鉴定猪肺炎支原体。在猪肺炎支原体的16SrRNA、P36和P463个基因内设计3个靶基因片段,用PCR标记Cy3探针,建立了用于检测和鉴定猪肺炎支原体的检测芯片。试验结果显示,猪肺炎支原体与检测芯片的3个靶基因（Mhp-16S、Mhp-P46和Mhp-P36）杂交呈阳性,猪鼻支原体只与Mhp-16S靶基因杂交呈阳性,其它所测细菌和病毒不与检测芯片的靶基因杂交。检测芯片的检测灵敏度和PCR相同,能达到10pg基因组DNA／50μL标记体系或反应体系。用检测芯片鉴定了3株疑似猪肺炎支原体临床分离株,结果有2株为猪肺炎支原体,1株为其它猪支原体。用检测芯片、P36-PCR和分离鉴定分别对45头病猪临床样品选择混合培养物中的猪肺炎支原体进行了检测,结果它们的检出率分别为20％（9／45）、22．2％（10／45）和8．9％（4／45）。检测芯片还检出有26．7％（12／45）的病猪感染了其它猪支原体。试验结果表明,所建立的检测芯片是一种快速检测和鉴定猪肺炎支原体的敏感特异性方法。     

猪肺炎支原体的培养和保存

我们从外地引进几株猪肺炎支原体标准株和流行株,并从慈溪分离出一株猪肺炎支原体,对其继续培养方法和菌株保存方法进行了一些工作,现小结如下。材料和方法1.猪肺炎支原体液体培养基我们参照中监所的培养基配方,配制了适合于支原体分离和培养的液体培养基,其组成成分为: 0.2%水解乳蛋白Hank’s液400ml 牛心消化液240ml     

内蒙古地区绵羊肺炎支原体的分离鉴定及序列分析

本试验无菌采取内蒙古地区某发病羊场的绵羊病变肺脏组织,接种于支原体液体培养基进行分离培养后获得1株支原体,根据分离株的培养特性、形态学观察及生化试验等,初步鉴定为绵羊肺炎支原体。然后提取分离株的基因组,用通用引物体外扩增出分离株16S rRNA序列,将该序列与GenBank中已知33种支原体序列进行比较,结果表明该序列与绵羊肺炎支原体标准株Y-98的16S rRNA序列的同源性为99%,鉴定该分离株为绵羊肺炎支原体。     

猪血清对猪肺炎支原体培养影响的研究

正一、材料(一)菌株1.猪肺炎支原体P-5722-3株,由美国硕腾动物保健公司提供,哈药集团生物疫苗有限公司保藏。2.猪肺炎支原体P-5722-3株CCU阳性标准品(CCU检测),由美国硕腾动物保健公司提供,哈药集团生物疫苗有限公司保藏。(二)完全培养基1.培养基组分A:根据猪肺炎支原体P-5722-3株培养基配方要求,制备组分A。2.培养基组分B:猪血清3.猪肺炎支原体P-5722-3株完全培养基由培养基组分A与培养基组分B共同制备,培养基组分A由哈药     

3种支原体培养基对猪肺炎支原体CJ株培养效果的比较分析

通过猪肺炎支原体CJ株接种3种猪肺炎支原体培养基后的生长活性研究发现,与其他2种培养基相比,接种改良Friis培养基培养2~3 d含菌量可达到1.0×109~10CCU/m L,具有生长迅速、含菌量高的特点,可用于猪肺炎支原体CJ株的培养。     

1株猪源肠道益生菌的分离鉴定及其生物学特性研究

从健康猪肠道中分离出1株对仔猪大肠杆菌病和副伤寒病病原有较强拮抗作用的菌株,命名为W-02,对其进行菌落形态、生理生化特性和16S rDNA系统发育分析,确定该菌株为凝结芽孢杆菌。对该菌株胃肠道耐受特性和安全性进行了研究,结果表明,W-02菌株在人工胃液、人工肠液、猪胆盐和80℃高温表现出很强的耐受能力,急性毒性试验结果表明该菌株为无毒菌株。     

Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine.

Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally.     

Mycoplasma hyorhinis in Taiwan: diagnosis and isolation of swine pneumonia pathogen

This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).     

A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas

An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas. Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes. Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface. Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study. The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy. Adherence of M. hyopneumoniae and M. flocculare and damage to the cilia were demonstrated using scanning electron microscopy. The pathogenic M. hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage. The non-pathogenic M. hyopneumoniae strain J did not adhere to mature cilia. Both M. flocculare strains Ms42 and 7160T adhered to mature and budding cilia. No obvious ciliary damage was observed with strain Ms42. Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T. This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia.     

Hematological and clinicochemical profiles of healthy swine and swine with inflammatory processes

The hematological and clinicochemical profiles of healthy swine and swine with inflammatory processes were investigated. Blood was collected at slaughter and postmortem examination was performed to select healthy swine and swine with pleuritis, pneumonia or abscesses. In healthy swine, the values of several variables revealed significant differences between gilts, barrows and boars. This was caused predominantly by the values obtained for boars. Inflammatory processes altered the values of most variables investigated, particularly for erythrocyte sedimentation rate, hemoglobin and hematocrit, for the activity of alkaline phosphatase, and for concentrations of iron, phosphate, albumin and fibrinogen in plasma. Compared with healthy swine, differences were largest for swine with metastatic abscesses and swine with both abscesses and other pathological lesions; differences were less pronounced in swine with solitary abscesses and were minor in swine with pneumonia and swine with pleuritis. Porcine hematological and clinicochemical profiles reflect the degree of inflammation.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Chlamydial infection and perinatal mortality in a swine herd

Chlamydia psittaci was believed responsible for an episode of high perinatal death loss in a swine herd in which 8.5 pigs per litter normally were weaned. In this episode, 18 sows produced 186 pigs, with 50 survivors. Chlamydia was found in tissue samples, and other bacterial or viral pathogens could not be identified. Chlamydia was diagnosed by isolation (ELISA), histologic examination using immunoperoxidase staining techniques, and electron microscopy. Previously, C psittaci has not been considered in the differential diagnosis of swine perinatal mortality.     

猪肺炎支原体黏附蛋白的研究进展

猪肺炎支原体是导致猪慢性肺炎的致病因子,其致病过程主要由表面黏附蛋白介导完成。综述了肺炎支原体表面黏附蛋白（P36、P46、P65、P97、P102、P110、P159和P216）的研究进展,为猪慢性肺炎的治疗和诊断研究奠定了基础。     

Estimation of the cost of pneumonia in swine herds

The data from studies of pneumonia, in which growth rate and feed conversion were measured, were examined with respect to the expected change in feed efficiency associated with a specific reduction in growth rate attributable to pneumonia in a swine herd. Information from 5 other studies was used to determine the expected reduction in growth rate that would be associated with the severity of pneumonia in the herd as determined by lesions recorded at slaughter. Together these calculations provide a reasonable estimation of the economic loss associated with pneumonia in a swine herd.