Hemagglutination and hemagglutination inhibition with Chuzan virus.

Chuzan virus agglutinated erythrocytes of several species of animals including bovine. The hemagglutinating (HA) activity against bovine erythrocytes was dependent on NaCl molarity and was expressed best at 0.6 M, but it was independent of pH and temperature. Three strains of Chuzan virus isolated from 2 cows and a pool of culicoides midges had indistinguishable HA antigenicity. All cattle infected with the virus developed high titers of hemagglutination inhibiting (HI) antibody which changed in parallel with neutralizing (NT) antibody titers. Correlation between HI and NT antibodies was very high and the antibodies persisted for one year or more. Therefore it was concluded that the HI test is applicable for survey of Chuzan virus infection among cattle in place of the NT test.     

Mycoplasma gallisepticum-induced alterations in chicken red blood cells

Incubation of Mycoplasma gallisepticum with washed chicken red blood cells for 1 hr or 5 hr resulted in altered red blood cell surface morphology and perforations of the cells.     

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.     

猪肺炎支原体对猪气管上皮细胞的黏附作用

采用气管结扎、链霉蛋白酶灌注冷消化法分离猪气管上皮细胞,并以对数生长期的猪肺炎支原体(Mhp)感染猪气管上皮细胞,通过间接免疫荧光技术定性定量研究不同滴度的猪肺炎支原体对猪气管上皮细胞的黏附及相同滴度的猪肺炎支原体随时间变化对猪气管上皮细胞的黏附。结果表明,分离的猪气管上皮细胞存活率为95%以上,广谱角蛋白染色阳性;Mhp野毒株XLW-1与猪气管上皮细胞37℃作用30min就能看到少量的支原体黏附,作用4h以上时黏附更明显,并且,二者作用1、2、4、6、10h,XLW-1对猪气管上皮细胞的黏附与阴性对照相比差异极显著(P0.01);1×107、2.5×106 CCU/mL的XLW-1与猪气管上皮细胞37℃作用6h,能看到支原体黏附于细胞表面,并且与阴性对照相比差异极显著(P0.01),而其他低滴度的XLW-1则看不到黏附。     

不同毒力的猪肺炎支原体对宿主细胞的黏附

为研究不同毒力的猪肺炎支原体对宿主细胞的黏附作用,本研究采用体外培养的猪气管环检测不同毒力的猪肺炎支原体代表菌株对猪气管上皮细胞及纤毛的损伤情况,初步验证不同猪肺炎支原体代表菌株的毒力.应用间接免疫荧光技术研究不同猪肺炎支原体分离株、标准株、168致弱株及疫苗株对猪肾细胞PK15、肺泡上皮细胞SJPL和猪肺泡巨嗜细胞3D4/31的黏附情况.结果表明猪肺炎支原体野毒株XLW-1能引起猪气管上皮细胞严重病变及纤毛损伤、脱落;国际标准弱毒株J株仅能引起猪气管上皮细胞轻微病变;168疫苗株既不能引起猪气管上皮细胞病变,也不能引起纤毛损伤.野毒株XLW-1、国际标准强毒株232株、弱毒株J株及168疫苗株均能黏附PK15、SJPL和3D4/31,168致弱株可以黏附PK15和SJPL.野毒株、标准强毒株、标准弱毒株、168致弱株及疫苗株均能黏附PK15、SJPL和3D4/31.     

In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells.

A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigen-reactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.     

Vaccination of sows against Mycoplasma hyopneumoniae with Hyoresp

The aim of the study was to investigate the serological reactions of pregnant sows to vaccination with Hyoresp. Further investigations were performed in the offspring of these sows to follow the dynamics of maternal antibodies and the reaction to vaccination at different points in time. The study was conducted in three farrow-to-finish herds endemically infected with M. hyopneumoniae. A total of 30 gilts and 31 sows were vaccinated 8 and 4 weeks ante partum with Hyoresp (Merial GmbH) or given phys. saline solution as a placebo. The offspring was divided into three groups receiving Hyoresp at 1 and 4 or at 4 and 8 weeks of age. The control group was treated with phys. saline solution at 1 and 4 weeks of age. Before vaccination, antibodies against M. hyopneumoniae were detected in 85% of the gilts and 68% of the sows, confirming the endemic infection of the herds. Vaccination of the sows induced a significant increase in the antibody concentration in serum within four weeks and enhanced the concentration of antibodies in the colostrum. As expected, significantly enhanced levels of antibodies were also detected during the first four weeks of life of the offspring of vaccinated sows. The piglets' serological reaction to vaccination at 1 and 4 weeks of age showed marked interferences with maternal antibodies, so that a reaction could be demonstrated only at 8 weeks of age. The serological reaction of piglets vaccinated at 4 and 8 weeks of age was much stronger than that of piglets vaccinated earlier. Surprisingly, the vaccination status of the sow had no effect on the serological response of the piglets in either vaccination scheme. Maternal antibodies are known to reduce the risk of M. hyopneumoniae infections in piglets. Vaccinating the sows against M. hyopneumoniae may thus be an option for farrowing-to-finish herds with an enhanced risk for infections due to ineffective separation of different age groups, poor gilt acclimatisation or high gilt replacement rates.     

H3N2亚型犬流感病毒血凝及血凝抑制试验的研究

为优化H3N2亚型犬流感病毒(CIV)的血凝抑制(HI)试验方法,本研究应用不同种类红细胞进行CIV的血凝(HA)试验,应用不同种类红细胞和不同血清处理方法进行CIV的HI试验,评价其对HA和HI试验的影响。结果表明,H3N2亚型CIV对鸡和犬红细胞的凝集性最好,对小鼠、猪和牛红细胞的凝集性较差。HI试验应用鸡红细胞悬液效果最好,受体破坏酶(RDE)和高碘酸钾处理可以有效去除犬血清中非特异血凝抑制素,但高碘酸钾对血清特异性抗体有损耗。本研究筛选出H3N2亚型CIV HI试验的最佳方法,为犬流感的血清学诊断提供技术支持。     

Interactions between the membranes of turkey cells and Mycoplasma meleagridis

We used Myoplsma meleagridis (MM) to infect the RP-9 cells and the eggshell membranes and scanning electron microscopy (SEM) and confocal microscopy to study the interactions between the organisms and the cell surfaces. The surface of the RP-9 cells contained numerous projections. After 24 hr of infection with MM, those projections were either lost or aggregated to the side; MM-like particles could be seen on the surface of the cells, and surface fluorescence could be detected by confocal microscopy. On the surface of MM-infected shell membranes were necrotic fibrous tissues and cells detected by SEM and an intense surface fluorescence detected by confocal microscopy. These results indicate that MM infection of the cell surface can result in cellular damage.     

Interactions of Mycoplasma hyopneumoniae membranes with porcine lymphocytes

Nonspecific mitogenicity of Mycoplasma hyopneumoniae membranes for blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) from swine was investigated. Additionally, the influence of respiratory tract exposure to the same membrane preparation on responsiveness of these cells was evaluated. Membranes utilized in lymphocyte transformation tests and for inoculation of swine were prepared by osmotic lysis of mycoplasma cells. Conventionally reared and cesarean-derived, colostrum-deprived swine were given membranes intratracheally and responses of BL and LNL to membranes were assessed from postinoculation day 0 to 14. Utilizing a stimulation index of 3 as the cutoff, heated (56 C) M hyopneumoniae membranes exerted moderate nonspecific stimulation of BL 11 of 12 times when BL were collected from normal (control or preinoculation) swine. Similarly, LNL from conventionally reared and control groups of swine were stimulated nonspecifically 4 of 6 times by the same membrane preparations. Exposure of the respiratory tract to membranes appeared to have no influence on stimulation responses of BL at postinoculation days 6 or 13, whereas moderate to marked increases in responsiveness of LNL were detected when collected at necropsy on postinoculation days 7 or 14. These findings indicated that compartmentalization of lymphocyte sensitization in the bronchial lymph nodes resulted from respiratory tract exposure to mycoplasmal membranes. Results obtained confirm that M hyopneumoniae has a moderate nonspecific stimulatory effect on porcine lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

环丙沙星在支原体肺炎猪体内的药动学

18头健康杜洛克×长白×大白杂交猪,分3组,每组6头,通过气管内接种含有猪肺炎支原体的病肺悬液复制疾病模型后,以5.0mg/kg静注、肌注及内服给药进行环丙沙星药物动力学研究.高效液相色谱法测定血浆中药物浓度.MCPKP药物动力学程序处理药时数据.结果显示感染猪静注给药的药时数据适合二室开放模型,t1/2α为0.59 h,t1/2β为3.52 h,Vd(area)为3.21 L/kg,ClB为0.645 L/(ks·h).感染猪肌注和内服给药后的药时数据则适合一级吸收一室模型,t1/2ka分别为0.09、0.31 h;tmax分别为0.46、1.41 h;Cmax分别为1.67、0.35 mg/L;F分别为97.30%、34.66%.上述结果表明,支原体性肺炎对环丙沙星静注给药在猪体内的分布有一定影响,但对其消除过程的影响不大;与健康猪比较,感染猪肌注给药的峰浓度、药时曲线下面积及生物利用度均显著提高,而内服给药的峰浓度、药时曲线下面积及生物利用度均显著降低.     

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.     

Oral challenge of turkey poults with Mycoplasma iowae

Day-old poults were inoculated orally each day for 7 days with 0.2 ml of Mycoplasma iowae, strain D112, 10(8) colony-forming units/ml. Cloacal swabs were taken from each poult during the inoculation period and at selected intervals until 21 days after the last inoculation. Most poults shed mycoplasmas persistently after inoculation. Cloacal swabs from eight out of ten poults were positive at 21 days after the last inoculation. Feces of poults in the infected group were normal, and there was no significant rise in cloacal temperature. At necropsy, mycoplasmas were recovered from tissues of the respiratory tract, gastrointestinal tract, spleen, and kidney. In the gastrointestinal tract, the most frequent recoveries were from the wall of the distal portion of the small intestine, cecum, and large intestine. Recovery of M. iowae from these organs and tissues indicated infection following oral challenge.     

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     

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.     

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.     

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.     

“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。