Adherence of Mycoplasma hyopneumoniae to cell monolayers

This work was an attempt to develop an in vitro adherence model for Mycoplasma hyopneumoniae, using monolayers of human and porcine lung fibroblasts and porcine kidney cells. Mycoplasma hyopneumoniae grown in Friis mycoplasma broth was radiolabeled with 35[S]-methionine, washed, concentrated, and inoculated on the monolayers. After 15 minutes of centrifugation to facilitate adherence, monolayers were washed 3 times, dissolved with 0.1N NaOH, and suspended in scintillation liquid, and the radioactivity was determined in a liquid scintillation counter. Adherence, measured as a percentage of counts added, varied according to the mycoplasma strain and the cell line used. Comparison of strains J, 144L, and 232 of M hyopneumoniae revealed 7.5 +/- 5.9, 31.9 +/- 13, and 9.6 +/- 5% adherence to porcine kidney cells, respectively. Slightly different, but proportionally the same relationships were obtained with swine or human fibroblasts. Adherence was decreased slightly by repeated washings of the mycoplasma-treated cell monolayers; however, a plateau was reached, indicating irreversibility of the adherence process. Pretreatment of cell monolayers with nonlabeled organisms substantially blocked adherence by labeled organisms. Dilution of labeled organisms resulted in an increased proportion adhering. Therefore, it appears that the adherence was a receptor-dependent event. Treatment of the mycoplasmas with trypsin prior to the inoculation of monolayers resulted in a marked reduction in adherence. Treatment of the mycoplasmas with hyperimmune swine serum against M hyopneumoniae or normal swine serum resulted in 80 to 90% reduction of adherence; however, no inhibition occurred when mycoplasmas were treated with purified IgG from the hyperimmune serum.     

Diagnosis of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae, the cause of enzootic pneumonia, remains an important pathogen in the swine industry. This small, complex organism colonizes the ciliated cells of the respiratory tract, resulting in little exposure to the immune system. Confirming the presence of M. hyopneumoniae, as well as identifying its role in respiratory disease and pneumonia, remains challenging to the veterinary profession. While culture of the organism remains the gold standard for identification, the use of serology, the polymerase chain reaction and various assays to detect the presence of M. hyopneumoniae in tissue is common in diagnostic laboratories. Because of the role M. hyopneumoniae plays in increasing the severity of pneumonia associated with concurrent bacterial and viral infections, understanding the pathogenesis and diagnostic assays available is critical for developing effective intervention strategies to control respiratory disease on a herd basis.     

猪肺炎支原体表面蛋白P46基因的克隆与序列比较

猪肺炎支原体(Mycoplasma Hyopneumoniae,Mhp)兔化弱毒株R659株、济南强毒株、强毒Z株、国际标准株232,通过A26培养基培养,提取DNA;利用PCR技术从四株猪肺炎支原体中均能扩增出目的条带.将该序列克隆到pGEM-T-Easy载体上测序.结果表明,克隆序列全长1 152 bp,编码383个氨基酸和一个终止子(TAA);该序列中含有3个TGA编码Trp,而不是终止密码子.比较兔化弱毒株与济南强毒株、国际标准株232及NCBI上发布的J株的P46基因序列,发现它们的同源性分别为98.6%、99.2%、99.2%;比较它们编码的氨基酸发现它们的同源性分别为98.7%、99.2%、99.2%.结果表明猪肺炎支原体的P46基因在猪肺炎支原体种内是很保守的,因此建立以P46蛋白为诊断抗原的ELISA具有潜在的意义.     

Morphological and ultrastructural studies of Mycoplasma flocculare and Mycoplasma hyopneumoniae in vitro.

Cells of Mycoplasma flocculare were found to vary in size and shape, especially in the later phases of growth, whereas those of Mycoplasma hyopneumoniae were fairly uniform irrespective of growth phases. Filamentous cells were present in cultures of M flocculare in the stationary and declining phases, but were never found in cultures of M hyopneumoniae. The filamentous and bizarre forms observed when mycoplasmas were suspended in phosphotungstic acid probably result from the action of the hypotonic solution. The surface of all cells was covered by a fuzzy coat consisting of fine hairs or bristles. An electron-lucent region was usually seen in cells negatively stained after centrifugation, but was only occasionally seen in cells negatively stained directly from the medium. Intracytoplasmic membranes were present in sectioned cells. No attachment organelle was found in cells of either species.     

PCR方法测定猪肺炎支原体培养物菌数

采用常规的CCU方法对猪肺炎支原体培养物菌数测定中存在很多的弊端，如耗时长、影响因素多等，难以用于实际生产中。本研究采用PCR方法能有效地克服CCU方法的缺点，具有重复性好、特异性强、快速准确等特点，为大规模猪肺炎支原体培养和疫苗生产提供保证。     

Experimental evaluation of Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge

The objective of this study was to evaluate the efficacy of a new Mycoplasma hyopneumoniae bacterin against a Korean M. hyopneumoniae challenge under experimental conditions. Fifteen pigs were allocated randomly into 3 groups (5 pigs per group) that were designated in 1 of 3 ways: vaccinated-challenged, unvaccinated-challenged, or unvaccinated-unchallenged. The pigs in the vaccinated-challenged group were immunized with an M. hyopneumoniae whole-cell bacterin at a 1.0 mL dose-level at 21 d old. At 42 d old (0 d post-challenge), the pigs in the vaccinated-challenged and unvaccinated-challenged groups were inoculated intranasally with a strain of Korean M. hyopneumoniae. Vaccinated-challenged pigs elicited a strong cell-mediated immunity as measured by M. hyopneumoniae-specific interferon-γ secreting cells when compared with unvaccinated-challenged pigs. Vaccination of pigs with this new M. hyopneumoniae bacterin reduced nasal shedding and lung lesions. The evaluated vaccine was therefore considered effective in controlling M. hyopneumoniae infection.     

猪支原体肺炎活疫苗（168株）肺内免疫机制研究

为研究猪支原体肺炎活疫苗(168株)的免疫机制,通过肺内接种免疫5 ～ 10日龄仔猪,并于免疫后不同时间点检测血清中IgG抗体效价、全血中淋巴细胞转化效率、呼吸道局部的IFN-γ浓度和特异性SIgA滴度,于免疫后28 d剖杀采集呼吸道上皮组织,通过扫描电镜法与原位杂交检测法观察疫苗株在呼吸道的存留以及对纤毛的影响情况.结果发现,免疫后猪血液中淋巴细胞转化增强1.52～2.01倍,支气管表面IFN-γ浓度和特异性SIgA滴度持续增加,但血清抗体一直未检测到.扫描电镜与原位杂交检测结果发现疫苗株能有效地黏附在支气管纤毛上皮细胞上,但对纤毛的影响较小.由此表明,猪支原体肺炎活疫苗(168株)通过肺内免疫可有效激活全身细胞免疫及呼吸道局部的黏膜免疫与细胞免疫反应,而且还可以通过黏附支气管纤毛上皮细胞产生占位效应而对上皮组织不产生损伤.     

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a ''one factor at a time'' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.     

Relationship between group B streptococcal serotypes and cell surface hydrophobicity.

Cell surface hydrophobicities of streptococci of serological group B were determined by the adherence of the bacteria to hexadecane droplets. A significant adherence to hexadecane was observed with the group B streptococcal type reference strains Ib, V, Ic, R and X, but not with those of serotype Ia, II, III and IV. Cultivation of the bacteria in microcapsule-inducing media reduced the hexadecane adherence properties. The adherence to hexadecane was not related to fibrinogen binding properties of the cultures. Screening a large number of group B streptococci isolated from humans and bovines revealed that those with polysaccharide type antigen alone were generally hydrophilic, those with protein antigen alone or with protein antigen in combination with polysaccharide antigen were mostly hydrophobic. Cultivation of the bacteria under microaerobic conditions or after a single mouse passage enhanced microcapsule production and correspondingly reduced the hexadecane adherence values. Treatment of the bacteria by guanidinium chloride or by neuraminidase enhanced the hexadecane adherence. The hydrophobic component on group B streptococcal surface appeared to be partly inactivated by heat or proteolytic treatment of the bacteria.     

猪肺炎支原体及其生物学研究进展

猪肺炎支原体是引起猪支气管肺炎的主要病原 ,严重危害着养猪事业的发展。虽然猪肺炎支原体的结构简单 ,但由于它对营养要求较高 ,故培养难度大 ,也是人们不能对其深入研究的一个主要原因。近年来利用克隆技术 ,对猪肺炎支原体从基因水平上进行了多种研究 ,已经取得了一些可喜成果。文章从它的生物学特性、荚膜结构、膜蛋白研究以及基因水平的研究等方面进行了综合性论述 ,并简明地指出了目前研究中存在的问题以及未来展望     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

猪肺炎支原体 MY-99株的致病性

将猪肺炎支原体MY-99株人工培养F100株经鼻腔接种于健康成华×大约克杂交猪,2周后60%的猪出现气喘,个别猪伴有轻微咳嗽;病理切片可见到支气管周围多量淋巴样细胞浸润、肺泡壁增厚等典型的间质性肺炎病变.试验猪平均日增重比对照猪显著降低,接种后0～14 d期间降低了31.05%(P<0.01),15～28 d期间降低了42.01%(P<0.01),29～42 d期间降低了0.1%(P>0.05).试验猪血清中的猪肺炎支原体抗体水平在2周后最高(1∶24.2),4周后降至1∶23.9,6周后最低(1∶22.5),显示猪肺炎支原体MY-99株诱发了急性猪地方流行性肺炎,同时从试验猪肺脏中分离出了猪肺炎支原体.超微切片显示,其菌体大小为0.33～0.48 μm,并缺乏细胞壁;革兰氏染色为阴性类球形菌体,在压力下可以通过0.45 μm的细菌滤膜,菌落呈现煎蛋状.     

猪支原体肺炎疫苗研究进展

猪肺炎支原体是引起猪支原体肺炎的病原,该病呈世界性流行,死亡率虽然不高,但由于长期性和消耗性的流行,使饲料转化率降低,并引起猪的多种并发病,是造成养猪业经济损失最重要的疾病之一.目前疫苗免疫是预防猪支原体肺炎和减少经济损失最有效的手段之一,猪支原体肺炎疫苗主要有灭活疫苗和减毒活疫苗.这些疫苗已得到广泛的应用,并在临床上...     

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.     

Control of Mycoplasma hyopneumoniae infections in pigs

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, occurs worldwide and causes major economic losses to the pig industry. The organism adheres to and damages the ciliated epithelium of the respiratory tract. Affected pigs show chronic coughing, are more susceptible to other respiratory infections and have a reduced performance. Control of the disease can be accomplished in a number of ways. First, management practices and housing conditions in the herd should be optimized. These include all-in/all-out production, limiting factors that may destabilize herd immunity, maintaining optimal stocking densities, prevention of other respiratory diseases, and optimal housing and climatic conditions. Strategic medication with antimicrobials active against M. hyopneumoniae and, preferably, also against major secondary bacteria may be useful during periods when the pigs are at risk for respiratory disease. Finally, commercial bacterins are widely used to control M. hyopneumoniae infections. The main effects of vaccination include less clinical symptoms, lung lesions and medication use, and improved performance. However, bacterins provide only partial protection and do not prevent colonization of the organism. Different vaccination strategies (timing of vaccination, vaccination of sows, vaccination combined with antimicrobial medication) can be used, depending on the type of herd, the production system and management practices, the infection pattern and the preferences of the pig producer. Research on new vaccines is actively occurring, including aerosol and feed-based vaccines as well as subunit and DNA vaccines. Eradication of the infection at herd level based on age-segregation and medication is possible, but there is a permanent risk for re-infections.     

“猪地方性肺炎及其防治控制”专栏四：猪肺炎支原体的防治措施

猪肺支原体是猪地方性肺炎的主要病原体,其普遍存在于世界各地,会给养猪业造成巨大的经济损失。此病原体感染猪后通常黏附于猪呼吸道纤毛上皮,并会造成纤毛上皮的损伤。猪感染后主要表现为慢性咳嗽症状、容易发生其他呼吸道感染和生产性能下降。该病的控制可通过多种措施实现,首先应该完善管理措施,改善畜舍饲养环境,这包括实行全出/全进的饲养方式、减少会破坏群体免疫水平的因素、维持最佳的饲养密度、防止其他呼吸道疾病的感染,以及提供最佳的畜舍及环境条件。其次,当猪群处于呼吸道疾病感染的威胁之下时,战略性使用能够有效地预防猪肺炎支原体和最好还能预防大多数继发感染细菌的药物对预防本病非常有效。最后,商用疫苗已被广泛地用来控制猪肺炎支原体感染,接种疫苗的优点在于其能够减少临床症状、减轻肺脏损伤、减少药物的使用和提高猪群的生产性能。但是,疫苗仅能提供部分保护作用,并且不能防止病原体在猪体内的定殖。因此,应根据猪群的种类、猪场的生产系统和管理体制、感染的类型及猪农的喜好选择不同的免疫策略（免疫时机、母猪免疫、免疫接种再结合抗菌素治疗）。新疫苗正在紧锣密鼓的研究之中,如气雾苗、通过饲料接种的疫苗以及亚单位苗和DNA疫苗。按年龄进行隔离饲养和药物治疗在猪群水平上根除猪肺炎支原体感染是可能的,但该病复发的威胁将永久存在。     

猪肺炎支原体的防治措施

猪肺支原体是猪地方性肺炎的主要病原体,其普遍存在于世界各地,会给养猪业造成巨大的经济损失.此病原体感染猪后通常黏附于猪呼吸道纤毛上皮,并会造成纤毛上皮的损伤.猪感染后主要表现为慢性咳嗽症状、容易发生其他呼吸道感染和生产性能下降.该病的控制可通过多种措施实现.首先应该完善管理措施.改善畜舍饲养环境,这包括实行全出/全进的饲养方式、减少会破坏群体免疫水平的因素、维持最佳的饲养密度、防止其他呼吸道疾病的感染,以及提供最佳的畜舍及环境条件.其次,当猪群处于呼吸道疾病感染的威胁之下时,战略性使用能够有效地预防猪肺炎支原体和最好还能预防大多数继发感染细菌的药物对预防本病非常有效.最后,商用疫苗已被广泛地用来控制猪肺炎支原体感染,接种疲苗的优点在于其能够减少临床症状、减轻肺脏损伤、减少药物的使用和提高猪群的生产性能.但是,疫苗仅能提供部分保护作用,并且不能防止病原体在猪体内的定殖.因此,应根据猪群的种类、猪场的生产系统和管理体制、感染的类型及猪农的喜好选择不同的免疫策略(免疫时机、母猪免疫、免疫接种再结合抗菌素治疗).新疫苗正在紧锣密鼓的研究之中.如气雾苗、通过饲料接种的疫苗以及亚单位苗和DNA疫苗.按年龄进行隔离饲养和药物治疗在猪群水平上根除猪肺炎支原体感染是可能的,但该病复发的威胁将永久存在.     

Immunological and pathological reactions in piglets experimentally infected with Mycoplasma hyopneumoniae and/or Mycoplasma flocculare.

The aim of this work was to examine in vivo whether infection with Mycoplasma hyopneumoniae (M. hyop) and/or Mycoplasma flocculare (M. floc) would interact and influence the severity of enzootic pneumonia in piglets. Specific pathogen-free, hysterectomy-derived piglets were allocated to six groups and experimentally inoculated with M. hyop. and/or M. floc at the age of 2 or 8 weeks. Clinical symptoms, frequency of coughing and temperature measurement were noted daily. Lung lesions were recorded by post-mortem examination and histological observations. The cross-inoculation with both mycoplasmas did not influence the clinical or the pathological picture of the disease. Evolution of specific and crossreacting antibodies was analyzed by ELISA and immunoblotting. Animals inoculated with M. floc did not develop any lesions but showed a weak antibody response 6-8 weeks post-infection (p.i.). No cross-reacting antibodies against M. hyop proteins were detected. In animals inoculated with M. hyop, the first antibody response was detectable 4-5 weeks p.i. and was stronger in piglets infected at the age of 2 weeks than at the age of 8 weeks. Three cross-reacting antibodies against M. floc proteins with molecular weights of 110, 47 and 33 kDa were detected by antibodies to M. hyop. Experimental infections with both mycoplasmas did not show differences in the pattern of species-specific proteins.