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.     

Expression of secreted and membrane-bound mucins in the airways of piglets experimentally infected with Mycoplasma hyopneumoniae

Immunohistochemistry was used to demonstrate secreted mucins MUC2, MUC5AC and MUC5B and membrane-bound mucin MUC4 in the pulmonary bronchioles of piglets experimentally infected with Mycoplasma hyopneumoniae. Conventional status, Landrace-Duroc cross-bred piglets, 13 days of age, were randomised to two groups. One group (n=20) was infected by the intra-tracheal route with the SNU98703 strain of M. hyopneumoniae, and a group of 12 animals acted as uninfected controls. Five infected and three uninfected piglets were euthanased on the day of infection and at 7, 21, and 35 days post-inoculation (PI). Membrane-bound MUC4 and secreted MUC5AC were the predominant mucins produced in the bronchioles of the piglets in response to M. hyopneumoniae infection, but by day 35 PI, all labelled mucins had returned to pre-infection levels, contemporaneous with reduced pulmonary lesion scores. The increased mucin production may result from direct stimulation of the epithelium by mycoplasmal infection, or may arise indirectly following M. hyopneumoniae-induced ciliostasis.     

Cytokine expression in porcine lungs experimentally infected with Mycoplasma hyopneumoniae

To gain further insight into the pathogenesis of porcine enzootic pneumonia (PEP), cytokine expression in different pulmonary compartments was examined. Mycoplasma hyopneumoniae (Mh) and proinflammatory and immunoregulatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-alpha) were detected by immunohistochemical methods in porcine lungs experimentally infected with Mh. Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 1- to 5-week post-inoculation (wpi). Three Mh-free pigs were taken as controls. Mh-antigen was shown in paraffin-wax-embedded tissues by immunohistochemistry in the luminal surface of bronchial and bronchiolar epithelial cells of all Mh-infected pigs. Significant increase in cytokine expression was detected on snap-frozen tissues from the bronchoalveolar exudate of the airways, mononuclear cells of the alveolar septa and macrophages and lymphocytes of the peribronchial and peribronchiolar lymphoid tissue, from 1 wpi onwards, compared to expression in non-pneumonic lungs. The main cytokines in the BALT of Mh-infected animals that showed an increase were IL-2, IL-4, IL-8, IL-10 and TNF-alpha. In the alveolar septa and bronchoalveolar exudate IL-1 (alpha and beta), IL-2, IL-4, IL-8 and IL-10 expression also increased in infected animals.     

Growth performance and whole-body composition of pigs experimentally infected with Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (Mh) is the primary infectious pathogen responsible for enzootic pneumonia in pigs. Although Mh is thought to impair growth performance, whole-body composition, and fat and protein accretion in pigs with pneumonia have not been reported and the mechanism through which Mh reduces growth is unknown. The objectives of this study were to evaluate the effects of Mh on growth performance, whole-body composition, and protein and fat accretion in nursery pigs and to determine whether Mh infection increases the expression of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha). Sixty-four 2-wk-old Mh-free pigs were used (two trials) in a randomized complete block design. In each trial, two pigs were housed in each of 16 disease-containment chambers. At 4 wk of age, pigs were inoculated intratracheally with 3 mL of Mh broth (P5722-3, 10(7) cfu/mL) or sterile Friis culture medium. Clinical signs of disease and feed intake were monitored daily and body weight was determined weekly for 4 wk. Whole-body composition was determined from pigs killed 0, 14, and 28 d after inoculation, and the comparative slaughter technique was used to estimate protein and fat accretion. At death, gross lung lesions were quantified, and lung tissue was collected to verify the presence or absence of Mh, and to determine cytokine mRNA levels. Control pigs displayed no overt signs of infection and were Mh-negative and free of pulmonary lesions. Pigs inoculated with Mh showed pneumonic coughing (P < 0.005), were Mh-positive, and had pulmonary lesions that affected 4.5% (P < 0.01) and 14.1% (P < 0.001) of total lung surface area at 14 and 28 d, respectively, after inoculation. Ribonuclease protection assays revealed increased IL-1beta (P < 0.04) and TNF-alpha (P < 0.06) mRNA in lung tissue collected from a lesion site compared with tissue collected 10 cm from a lesion site or from control pigs. Interestingly, Mh did not depress weight gain or feed efficiency during any week of the 28-d study (P > 0.10). Moreover, Mh did not affect whole-body fat or protein accretion (P > 0.10). Thus, in spite of inducing disease and expression of inflammatory cytokines, Mh alone did not affect growth performance and whole-body composition of nursery pigs during the 4-wk experiment. The ability of pigs to contend with Mh may have resulted from the absence of other pathogens that generally co-exist with Mh under commercial conditions.     

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae.

Seven hysterectomy derived piglets were repeatedly challenged with Mycoplasma hyoneumoniae during the first week of life. Samples of trachea, bronchi and lung tissue collected 2–11 weeks post-inoculation (p.i.) were examined using light and electron microscopy. Autoradiography was used to study in more detail the site of M. hyopneumoniae multiplication. Gross lesions were observed in lung tissue and were characterized by hyperplasia of the epithelium and an increased mononuclear cell accumulation in perivascular and peribronchiolar areas. Mild lesions of the trachea and the bronchi, including epithelial hyperplasia and infiltration of the lamina propria by inflammatory cells, were noted.

Electron microscopy showed that, 2–6 weeks p.i., changes in the mid-trachea and bronchi surface consisted of the loss of cilia. Mycoplasmas covered tufts of cilia remaining on the epithelial cell surface. Scanning and transmission electron micrographs showed that they were predominantly found closely associated with the top of cilia. No specialized terminal structure could be seen and no mycoplasma cells were identified lying free in the lumen nor in close contact with the plasma membrane of cells of microvilli. Some fine fibrils radiating from one mycoplasma to another or to cilia were seen at higher magnification by scanning electron microscopy. Six to eleven weeks p.i., a disrupted epithelial surface lacking cilia was observed. Cells were desquamated and shed into the lumen with cellular remains containing droplets of mucus.

Autoradiography revealed that label corresponded to the observed mycoplasma distribution. At the top of cilia, a high density of labeling was visible in the zone of high mycoplasma concentration. Therefore, incorporation of the label in the mycoplasma is proof or their multiplication in the trachea.

The intimate association between the mycoplasma and cilia may be an important factor in the pathogenesis of the disease caused by M. hyopneumoniae (swine enzootic pneumonia).     

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.     

An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia (EP), a highly prevalent respiratory disease that affects pigs worldwide. Previous studies have demonstrated that M. hyopneumoniae infection can be longer than 185 days; however, the total duration of infection has not been determined yet. Therefore, the objective of this study was to determine the duration of M. hyopneumoniae infection in asymptomatic carriers. To achieve our goal, 60 pigs were inoculated with M. hyopneumoniae strain 232 and the persistence of M. hyopneumoniae in the respiratory tract was assessed by detection of the bacterial DNA in bronchial swabs and the ability of the infected pigs to transmit the pathogen to sentinels. Infection of the inoculated animals was demonstrated by the detection of M. hyopneumoniae DNA in nasal swabs, seroconversion to the bacteria and the onset of dry coughing. Experimentally infected pigs shed M. hyopneumoniae prior to and after the cough was observed. M. hyopneumoniae DNA was detected in 100% of experimentally infected pigs at 94 days post infection (dpi), 61% at 214dpi and 0% at 254dpi. Experimentally infected pigs transmitted the bacteria to sentinels at 80 and 200dpi. Results of this study have demonstrated that M. hyopneumoniae infected pigs can be incubatory as well as convalescent carriers of the pathogen and that convalescent carriers can remain infectious for up to 200 days. Total clearance of M. hyopneumoniae in the group was evidenced, demonstrating that duration of M. hyopneumoniae infection lasts less than 254 days.     

猪肺炎支原体的分离及PCR鉴定

猪喘气病(Mycop lasm a pneum on iae of Sw ine,M PS)是由猪肺炎支原体(Mycop lasm a hyopneum o-n iae,M hp)引起的慢性接触性呼吸道疾病,主要症状为咳嗽和气喘,以高发病率和低死亡率为特点。如果与其他肺部病原体(如猪繁殖与呼吸综合征病毒、放线杆菌、多杀性巴氏杆菌、猪流     

Chronologic localization of mycoplasma hyopneumoniae in experimentally infected pigs

The chronologic localization of Mycoplasma hyopneumoniae was examined by in situ hybridization in experimentally infected pigs for a period of 35 days after intratracheal inoculation. M. hyopneumoniae DNA was detected in bronchial and bronchiolar epithelial cells from infected pigs at 7, 14, 21, and 28 days postinoculation (DPI) and in alveolar and interstitial macrophages and type I pneumocytes from infected pigs at 14, 21, 28, and 35 DPI. Strong hybridization signals for M. hyopneumoniae were detected mainly at the luminal surface of bronchial and bronchiolar lining epithelial cells. When a hybridization signal was detected at the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole also exhibited peribronchiolar lymphoid cuffing. These observations suggested that the presence of M. hyopneumoniae in different tissues could be due to a difference in the duration of the infection.     

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.     

Detection of rotavirus in experimentally infected piglets.

Scouring and vomiting was induced in piglets by experimental infection with a field strain of rotavirus. Virus or viral antigen was detected in the small intestine by the fluorescent antibody technique and virus could also be demonstrated in infected tissue culture cells by immuno-fluorescence. Progeny particles in the epithelial layer of the small intestine were identified by electron microscopy. Three morphological types could be distinguished, often associated with electron-dense inclusion, and infected cells, though small in number, were present throughout the length of the small intestine.     

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YN200901株的分离鉴定

采集疑似猪肺炎支原体病理变化的肺脏,接种到改良牛心培养基、猪胃消化液培养基进行培养,经瑞特染色、镜检,菌落狄氏染色、PCR鉴定及序列比对、生化试验鉴定,抗猪肺炎支原体血清生长抑制试验,分离获得一株猪肺炎支原体云南地方流行菌株,命名为YN200901。试验结果为云南本地株猪肺炎支原体的后续研究提供了物质基础。     

猪肺炎支原体S株的分离与鉴定

从典型猪肺炎支原体病变肺组织传代分离到一株支原体,经培养特性、血清学鉴定、生化鉴定、PCR检测及测序分析证明其为猪肺炎支原体,纯化后命名为S株。将S株P46基因和P97基因R1区的氨基酸序列与其他菌种的相应序列进行同源性分析,该菌株P46基因氨基酸序列与其他菌种同源高达99%以上,P97基因R1区的氨基酸重复数为11个,不同于其他菌株;免疫原性试验结果表明该菌株具有良好的免疫原性。     

Treatment of pigs experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida, and Actinobacillus pleuropneumoniae with various antibiotics.

The authors have performed a comparative study of the efficacy of various in-feed medications for the treatment of 5- to 6-week-old specific pathogen-free (SPF) piglets experimentally infected on day 1 with Mycoplasma hyopneumoniae, on day 8 with Pasteurella multocida (serotype A), and on day 15 with Actinobacillus pleuropneumoniae (serotype 2). The treatment started on day 9 and continued for 12 consecutive days, then the piglets were euthanized for examination of macroscopic, histologic, and pathologic lesions and for the presence of mycoplasmas and bacteria in the lungs. Based on the results of clinical observations (respiratory signs, rectal temperature, body weight gain, and feed conversion efficiency), macroscopic and histologic lesions of the lungs, and microbiologic findings, the best results were obtained by treatment of pigs with Econor + chlortetracycline, followed by Tetramutin, Pulmotil, Cyfac, and lincomycin + chlortetracycline.     

Genotype distribution of Mycoplasma hyopneumoniae in swine herds from different geographical regions

Genetic heterogeneity of Mycoplasma hyopneumoniae in pigs has been reported, however there has been limited reproducibility on the molecular methods employed so far. The aim of this study was to modify and standardize a high-resolution multiple locus variable number tandem repeat analysis (MLVA), to investigate the genetic variability of M. hyopneumoniae circulating in the United States of America (USA), Brazil, Mexico and Spain. The MLVA was standardized on the basis of the number of tandem repeats in two Mycoplasma adhesins, P97 and P146, which are proteins involved in the adherence of the pathogen to cilia. A total of 355 samples obtained from the four countries were analyzed. The Simpson's diversity index for the assay was D = 0.976 when samples from all countries were combined. A large number of MLVA types (n = 139) were identified, suggesting that multiple M. hyopneumoniae variants are circulating in swine. The locus P97 had 17 different types with 2–18 repeats. The P146 locus showed higher heterogeneity, with 34 different types, ranging from 7 to 48 repeats. MLVA types that presented more than 30 repeats in P146 were found in Spain and Brazil, while shorter repeats were observed in the USA and Mexico. This simplified MLVA method proved to be an efficient tool for typing M. hyopneumoniae with a high degree of stability, repeatability, and discriminatory power. In conclusion, M. hyopneumoniae showed a high variable number tandem repeat heterogeneity and this assay can be applied in molecular epidemiology investigations within farms and productions systems.     

鸭源鸡杆菌对SPF雏鸡的感染特性研究

为研究鸭源鸡杆菌的流行病学特性,本研究采用从自然病例分离得到的鸭源鸡杆菌人工感染4日龄SPF雏鸡,通过形态学、PCR、荧光定量PCR和组织学等方法分别对感染后SPF雏鸡的组织脏器进行了细菌分离、检测和鉴定,用ELISA对其血清抗体水平进行检测.结果表明,人工感染鸡无临床症状,组织学检查感染鸡的肝脏、气管和肺脏组织均有损伤.人工感染后第3d和同居感染第2d鸡体内可分离出鸭源鸡杆菌,人工感染后第96d仍能够分离到鸡杆菌.ELISA方法证实人工感染后47 d和同居后32 d出现抗体高峰,持续2～3周,人工感染组和同居组抗体水平均高于对照组(p＜0.05);荧光定量PCR检测病料结果显示人工感染组和同居组的气管组织中细菌含量最高.本研究表明雏鸡在4日龄即可感染鸭源鸡杆菌,并能通过同居传播,长期带菌、排菌;感染后抗体产生缓慢、持续期短.本研究为鸭源鸡杆菌的流行病学、致病机理研究及防治措施的制定等提供了参考依据.     

人工感染空肠弯曲菌SPF雏鸡的病理学研究

空肠弯曲菌（campylobacter jejuni）是近十几年来被认识的世界范围广泛流行的人兽共患病原菌，它不仅能引起鸡的肠炎和肝炎，而且引起人类急性肠炎和食物中毒，英美流行病学研究已证实弯曲菌病作为食源性传染病的重要意义，因此本菌在公共卫生学上具有重要意义，