Comparison of Mycoplasma hyopneumoniae strains by serologic methods

Six field strains of Mycoplasma hyopneumoniae isolated from pneumonic lungs of pigs, reference strains 11 and J of M hyopneumoniae, Ms 42 strain of Mycoplasma flocculare, and BTS 7 strain of Mycoplasma hyorhinis were compared serologically, using hyperimmune antisera produced in rabbits. All strains of M hyopneumoniae were closely related as determined with the disk growth-inhibition test; however, differences in zone sizes indicated that some antigenic heterogeneity existed. Cross-reactions were not detected between M hyopneumoniae, M flocculare, and M hyorhinis with the growth-inhibition test. The metabolic-inhibition test was more useful for detection of intraspecies antigenic difference than was the growth-inhibition test, since antigenic diversity was clearly detected among M hyopneumoniae strains. Slight cross-reactions were observed between M hyopneumoniae and M flocculare. Using 2-dimensional immunoelectrophoresis, antigenic differences were observed among M hyopneumoniae strains, although many common components also were detected in electropherograms. Mycoplasma flocculare possessed a close antigenic relationship to M hyopneumoniae, as determined by two-dimensional immunoelectrophoresis, whereas both organisms were less related to M hyorhinis. Evidence obtained in this study indicated that strains of mycoplasmas tentatively identified as M hyopneumoniae were similar antigenically, but evidence was obtained also of some diversity in antigenic structure among these strains.     

Effect of growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine

In an attempt to develop better methods for consistent induction of pneumonia in naturally born swine, using cultures of Mycoplasma hyopneumoniae, fifty 6-week-old, naturally born pigs from a respiratory disease-free herd were used in 3 trials. Pigs inoculated with Mycoplasma hyopneumoniae strain 232 (passage 21) grown for 1 passage or 5 passages in Eagle minimal essential medium plus 20% porcine serum, with or without human lung fibroblasts, had a mean (+/- SD) value range between 5.4 +/- 3.6 and 9.2 +/- 2.1% of consolidated lung area. In the second trial, pigs inoculated 1, 2, or 3 days in succession with strain 232 grown in Eagle medium or Friis mycoplasmal medium with 20% porcine serum had between 5.1 +/- 7 and 8.7 +/- 4.3% of consolidated lung area. In the third trial, virulence of Mycoplasma hyopneumoniae strains 144L (p27), 11 (p26), J (p60), and 232 (p27) grown in Friis mycoplasmal medium was compared. Pigs inoculated with those strains had 5.1 +/- 4.1, 2.6 +/- 3.1, 0, and 4.3 +/- 4% of consolidated lung area, respectively. Significant differences were not found in consolidated lung area among groups in trials 1 and 2, and among groups of pigs inoculated with M hyopneumoniae strains 144L, 11, and 232 in trial 3. Pneumonia was not detected in pigs inoculated with strain J in trial 3.     

Cell-mediated and humoral immune response to Mycoplasma hyopneumoniae in pigs enhanced by dextran sulfate

The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs.     

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.     

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.     

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)     

Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3-10 and 4-7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.     

Diversity of Mycoplasma hyopneumoniae in pig farms revealed by direct molecular typing of clinical material

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia in swine. Various reports indicate that different strains are circulating in the swine population. We investigated the variety of M. hyopneumoniae strains by a newly developed genetic typing method based on the polyserine repeat motif of the LppS homolog P146. PCR amplification using M. hyopneumoniae specific, conserved primers flanking the region encoding the repeat motif, followed by sequencing and cluster analysis was carried out. The study included strains isolated from different geographic regions as well as lysates from lung swabs from a series of pig farms in Switzerland. High diversity of M. hyopneumoniae was observed but farms being in close geographic or operative contact generally seemed to be affected by the same strains. Moreover, analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain. The results indicate that multiple strains of M. hyopneumoniae are found in the swine population but that specific strains or clones are responsible for local outbreaks. The method presented is a highly reproducible epidemiologic tool allowing direct typing of M. hyopneumoniae from clinical material without prior isolation and cultivation of strains.     

A longitudinal study of the diversity and dynamics of Mycoplasma hyopneumoniae infections in pig herds

A longitudinal study was carried out to investigate the diversity and persistence of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains in four infected pig herds. In each herd, 20 pigs were randomly selected and blood and/or bronchoalveolar lavage (BAL) fluid was collected at 6, 10, 14 and 26 weeks of age. In the BAL fluid, quantitative PCR and MLVA (multiple-locus variable number of tandem repeats (VNTR) analysis) testing were performed for detection and typing of M. hyopneumoniae strains, respectively. At 26 weeks of age, the prevalence and severity of lung lesions were recorded at slaughter (minimum 50 pigs belonging to the same batch as the investigated pigs). The percentage of pigs testing positive on qPCR increased from 35% at 6 weeks to 96% at 26 weeks of age. With MLVA testing, positive pigs were found from 14 weeks onwards. Within each herd, only one distinct strain was detected, although clonal variants were identified in two herds. In three of the herds, the strain remained present until slaughter age. The percentage of pigs with Mycoplasma-like lesions ranged from 38% to 98%, and the average pneumonia score ranged from 1.7 to 11.9, respectively. The present field study documented that within a herd, mainly one distinct M. hyopneumoniae strain was present that persisted in the same animals for at least 12 weeks. This implies that the immune response of the animals following infection is not able to rapidly clear the infection from the respiratory tract.     

Variable number of tandem aminoacid repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae strains

The Mycoplasma hyopneumoniae genome contains at least 22 regions with a variable number of tandem nucleotide repeats (VNTRs) within coding DNA sequences (CDSs). In this work, the VNTR-containing CDSs were analysed in order to evaluate their degree of variation, possible correlations with antigenic properties, and their potential to be used as a basis for a strain typing PCR assay. We have analysed the VNTRs in five M. hyopneumoniae strains (J, 7448, 7422, PMS, and 232), based on published genomic sequences and on amplified and sequenced DNA segments. These VNTRs are distributed among 12 genes, most of which encode putative surface proteins, including known adhesins. The number of repeat units in any of the VNTRs is highly variable among the analysed strains, but they are, without exception, translationally in frame, and, therefore, code for a variable number of aminoacid repeats (VNTARs). These VNTARs determine putative structural, physicochemical and antigenic variations in the corresponding proteins, with potential implications for aspects associated to M. hyopneumoniae pathogenicity, such as cell adhesion and interactions with the host immune system. Considering that the characterized VNTARs are relatively stable, at least in vitro, and their sizes are strain-specific, we have developed a VNTR-based PCR assay for M. hyopneumoniae strain identification, useful for enzootic pneumonia (EP) diagnosis, strain typing, and distinction of circulating field isolates from vaccine strains in animals vaccinated against EP.     

Induction of enzootic pneumonia in pigs by the administration of an aerosol of in vitro-cultured Mycoplasma hyopneumoniae

The aim of this study was to establish whether enzootic pneumonia could be induced reliably in piglets by administering an aerosolised culture of Mycoplasma hyopneumoniae. Groups of five M hyopneumoniaefree Landrace x Large White piglets weaned at 11 to 14 days of age were exposed to aerosols of in vitro cultures of a virulent strain of M hyopneumoniae. In three separate trials, 14 of 15 pigs exposed to the bacteria developed pneumonia, but pigs exposed to the culture medium alone did not develop the disease. Lung pathology, both gross and histological, indicated acute disease. Ten of the pigs were tested for seroconversion by Western blot and they were all positive. The growth rates of the infected pigs were significantly reduced and the water consumption of the infected groups was also depressed. M hyopneumoniae was recovered from eight of the 15 infected pigs.     

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。     

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.     

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.     

Evaluation of virulence of Mycoplasma hyopneumoniae field isolates

The course of enzootic pneumonia, caused by Mycoplasma hyopneumoniae, is strongly influenced by management and housing conditions. Other factors, including differences in virulence between M. hyopneumoniae strains, may also be involved. The aim of this study was to evaluate the virulence of six M. hyopneumoniae field isolates and link it to genetic differences as determined by randomly amplified polymorphic DNA (RAPD) analysis. Ninety, conventional M. hyopneumoniae-free piglets were inoculated intratracheally with the field isolates, a virulent reference strain or sterile culture medium. Animals were examined daily for the presence of disease signs and a respiratory disease score (RDS) was assessed per pig. Twenty-eight days post infection, pigs were euthanized, blood sampled and a lung lesion score was given. Lung samples were processed for histopathology, immunofluorescence testing for M. hyopneumoniae and isolation of M. hyopneumoniae. RAPD analysis was performed on all M. hyopneumoniae strains. Significant differences between isolates were found for the RDS, lung lesion score, histopathology, immunofluorescence and serology. Based on the results of the different parameters, isolates were divided into three "virulence" groups: low, moderately and highly virulent strains. Typically, a 5000 bp RAPD fragment was associated with the highly and moderately virulent strains whereas it was absent in low virulent strains. It was concluded that high variation in virulence exists between M. hyopneumoniae strains isolated from different swine herds. Further studies are required to determine whether the 5000 bp fragment obtained in the RAPD analysis can be used as a virulence marker.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. II. Enzootic pneumonia of pigs: microbiological findings and their relationship to pathomorphology

Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems.     

ISOLATION OF MYCOPLASMA HYOPNEUMONIAE AND ITS ASSOCIATION WITH PNEUMONIA OF PIGS IN AUSTRALIA

Nine strains of mycoplasmas were isolated from the lungs of 5 pigs with clinical signs of naturally acquired enzootic pneumonia. Mycoplasma hyopneumoniae was isolated from the lungs of 1 pig and M. hyorhinis from the lungs of 4. An unidentified mycoplasma, which utilized arginine, grew rapidly in broth culture and produced centred colonies on solid medium, was isolated from the lungs of 4 pigs. The pathogenicity of the isolated strain of M. hyopneumoniae was determined by inoculation of pigs from an enzootic pneumonia-free herd. Enzootic pneumonia was produced in the lungs of all 5 pigs inoculated intranasally and intratracheally with broth cultures of the organism isolatied by limit dilution techniques. Enzootic pneumonia was produced in 3 of 6 pigs inoculated intranasally and intratracheally with M. hyopneumoniae purified by the passage of colonies on agar blocks. M. hyopneumoniae was isolated in pure culture from the lungs of all pigs with induced pneumonic lesions.     

Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method

The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs.     

Effects of Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine

Alveolar macrophages were collected at necropsy from pigs inoculated with Mycoplasma hyopneumoniae or Actinobacillus pleuropneumoniae or both and were tested for phagocytic capabilities, using in vitro techniques. Macrophages from noninoculated littermates were used as controls. Alveolar macrophages from pigs inoculated with either M hyopneumoniae or A pleuropneumoniae had significantly (P less than 0.05 to P less than 0.0025) higher phagocytic capacity than that of noninoculated controls. Macrophages from A pleuropneumoniae-inoculated pigs were comparatively more stimulated than were those from M hyopneumoniae-inoculated pigs. Pigs inoculated with M hyopneumoniae and then challenge-exposed with A pleuropneumoniae 2 and 4 weeks later had greatly reduced phagocytosis. Infection with M hyopneumoniae or A pleuropneumoniae caused stimulation of alveolar macrophage functions, and M hyopneumoniae infections may have suppressed phagocytic responses when pigs were challenge-exposed with a secondary pathogen (A pleuropneumoniae). This potential suppression may represent a prediposition of the host by M hyopneumoniae to secondary bacterial infections.