Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4–7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5–9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.     

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

Mycoplasma hyopneumoniae (M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio (R_n). This R_n-value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates.

Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R_n-values of the highly virulent and the low virulent isolates were estimated to be 1.47 (0.68–5.38) and 0.85 (0.33–3.39), respectively. No significant difference between the groups was found (P = 0.53). The overall R_n was estimated to be 1.16 (0.94–4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae, assessed for the first time by a reproduction ratio, shows that one piglet infected before weaning will infect on average one penmate during the nursery phase.     

A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds

Sixteen litters of seven pigs from each of nine Danish farrow-to-finish herds were followed to investigate the serological patterns caused by natural infection with Mycoplasma hyopneumoniae, Pasteurella multocida toxin and Actinobacillus pleuropneumoniae serotypes 2, 5–7, 12. In seven of the herds, pigs were followed as two separate cohorts started 4 weeks apart, and in two herds only one cohort was followed.

A total of 999 pigs were included in the study. The pigs were blood sampled at weaning and subsequently every fourth week until slaughter. All pigs were examined for antibodies against M. hyopneumoniae (enzyme-linked immunosorbent assay), P. multocida toxin (enzyme-linked immunosorbent assay) and A. pleuropneumoniae serotypes 2, 5–7, 12 (complement-fixation tests). The most-common pattern (28%) of seroconversion was that of pigs first seroconverting to A. pleuropneumoniae serotype 2, followed by seroconversion to M. hyopneumoniae. Each herd had a dominant serotype of A. pleuropneumoniae to which most pigs seroconverted. Seroconversion to the respiratory pathogens occurred mainly in the growing-to-finishing units (8–24 weeks). The risk of seroconversion to the P. multocida toxin was very low (<20%) and occurred late.

None, four and seven herds tested seropositive to PRRS and to swine influenza virus subtypes H3N2 and H1N1, respectively, when testing 10 pigs per herd (selected randomly among the study pigs) at the age of 20 weeks.     

Intranasal infection of ferrets (Mustela putorius furo) with canine parainfluenza virus.

Immunocompetent and cyclophosphamide-immunosuppressed ferrets were intranasally infected with canine parainfluenza virus (CPIV) and observed for clinical signs, histopathologic lesions, the immunocytochemical demonstration of CPIV antigen in the respiratory tract and scanning electron microscopic alterations of the tracheal epithelium until 36 days post infection (p.i.). In both groups, clinical signs were minimal, restricted to the upper respiratory tract and consisted of cough elicited by tracheal compression between 3 and 7 days p.i. Microscopically, inflammatory and degenerative lesions were observed in the trachea and less frequently in the nasal cavity; bronchiolitis or interstitial pneumonia was not demonstrated. By immunocytochemistry, CPIV antigen was demonstrated in tracheal epithelial cells, whereas nasal cavity, bronchi, bronchioles and lung were devoid of viral antigen. Ferrets given CPIV alone developed a minimal lymphocytic tracheitis with minimal loss of cilia and CPIV antigen was observed only 4 days p.i. 17 days p.i., normal epithelial organization and ciliary reappearance was reestablished. Ferrets treated with cyclophosphamide and infected with CPIV exhibited mild to moderate histological lesions as above with similar scanning electron microscopic changes until 36 p.i. Tracheal lesions consisted of intraepithelial and submucosal infiltration of lymphocytes and macrophages, focal epithelial hyperplasia and multifocal loss of cilia. In addition, mild and transient neutrophilic infiltration was observed. In immunosuppressed ferrets, viral antigen expression was prominent and demonstrated 4 and 8 days p.i. These data suggest that ferrets are susceptible to aerosol CPIV infection.     

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97 kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97 kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

Duration of Mycoplasma hyopneumoniae Infection in Gnotobiotic Pigs

Sixteen gnotobiotic pigs raised in flexible plastic isolators (four pigs per isolator) were inoculated with a culture of Mycoplasma hyopneumoniae. One pig was killed and underwent necropsy at weekly intervals for the following 16 weeks. Macroscopic lesions were observed in the lungs of 13 of 16 pigs and microscopic lesions were found in 14 of 16 pigs. Mycoplasma hyopneumoniae was cultured from the trachea or lungs from 10 of the 16 pigs. Scanning electron microscope studies showed areas of damage to the cilia, collections, of leucocytes and mucus, and mycoplasma in the trachea as well as the bronchi. These conditions were found in all the pigs seen at necropsy from nine to 16 weeks postinoculation and there was no evidence of noticeable regression or recovery during this 16 week period.     

Indirect ELISA for the detection of antibodies against Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1790) in foxes

Serological investigations focused on the detection of specific opisthorchiid liver fluke antibodies in silver foxes (Vulpes vulpes fulva). Animals were experimentally infected with Opisthorchis felineus (nos. 1 and 2) and Metorchis bilis (nos. 3–8) by feeding fish with a counted number of metacercariae. Four foxes remained as non-infected negative controls (nos. 9–12). For the indirect ELISA, an excretory–secretory antigen was produced by in vitro cultivation of O. felineus and M. bilis adults isolated from livers of experimentally infected hamsters.

Immunoglobulin G (IgG) seroconversion against homologous antigen took place between weeks 2 and 6 postinfection (p.i.) and foxes remained seropositive up to the end of the trial at week 41 p.i. In contrast, IgG titres against heterologous antigen remained significantly lower and stayed near the cut-off. All infected animals excreted opisthorchiid eggs, starting between weeks 2 and 4 p.i. The number of liver flukes found at necropsy was relatively low, except in one fox that was sacrificed at the week 11 p.i. These results suggest that the ELISA is a suitable tool for the detection of specific O. felineus and M. bilis antibodies in the fox.     

Scanning electron microscopic study of the lower respiratory tract in calves and adult cattle

The surface characteristics of the lower respiratory tract of two groups of cattle were studied with the scanning electron microscope. Group A comprised six one-week-old calves and group B four adult cows. None of the animals had overt respiratory disease or gross morphological evidence of pulmonary lesions. The trachea, bronchi, bronchioles and alveoli of the cranial and the caudal lobes of the right lung were examined. In both groups the luminal surface of the trachea and large bronchi were completely covered by cilia, apparently forming an efficient mucociliary escalator. In the adult animals there were some patchy areas in the trachea and large bronchi devoid of ciliated cells; these were considered abnormal. In the bronchi, non-ciliated cells, mainly mucus-secreting, were not easily identified unless they were discharging secretion. In small bronchi, non-ciliated cells were more evident and easily seen. The bronchioles had many non-ciliated cells and very few ciliated cells capable of forming a complete carpet for a mucociliary escalator. Types 1 and 2 alveolar epithelial cells and alveolar macrophages were identified in both groups. Pores of Kohn were found in the alveolar walls in all animals.     

Primary ciliary dyskinesia in a Staffordshire bull terrier

Primary ciliary dyskinesia (PCD) is a diverse group of inherited structural and functional abnormalities of the respiratory and other cilia, which results in recurrent respiratory tract infections. Primary ciliary dyskinesia was diagnosed in a 14-week old Staffordshire bull terrier that had a history of respiratory disease from 7 weeks of age. Pneumonia was diagnosed on thoracic radiographs and transtracheal aspirate. Transmission electron microscopy of the bronchi and trachea indicated the presence of both primary and secondary ciliary dyskinesia. The most prominent primary defects consisted of absent inner dyneim arms, absent radial spokes and absence of the central microtubules. These defects accounted for 62% of the total number of cross-sections screened. Non-specific ciliary abnormalities encountered most often were compound cilia, swollen cilia, addition/deletion of peripheral doublets and disorganised axonemes (26%). To the authors' knowledge, this is the first case of PCD described in the Staffordshire bull terrier and the first report of PCD in South Africa.     

Insertion sequence profiling of UK Mycoplasma bovis field isolates

Mycoplasma equigenitalium and Mycoplasma subdolum have been associated with infertility, endometritis, vulvitis and abortions in mares, and with reduced fertility and balanoposthitis in stallions. Despite their role in equine genital disorder, determinants of virulence and pathogenesis as well as factors provoking specific host immune responses have not been identified, so far. To establish the major immunogenic components of Mycoplasma (M.) equigenitalium and M. subdolum, antigen profiles of their type strains as well as 30 clinical isolates were compared by SDS-PAGE and immunoblot analysis using hyperimmune rabbit sera and equine sera from clinical cases. To define the major protein antigens of both mycoplasma species, detergent-phase fractionation with Triton X-114 was performed. Western blot analysis of 30 clinical isolates revealed a high similarity of their overall antigen profile with only slight differences. In contrast, monospecific polyclonal antibodies raised against detergent-phase proteins of the two mycoplasma species identified three prominent proteins (pST17, pST42, and pET45) undergoing variation in expression and size among clinical and clonal isolates.     

发根农杆菌介导的白花草木樨毛状根转化体系的建立

白花草木樨（Melilotus albus Medic.ex Desr.）是草木樨最常见的栽培用种之一，具有较强的抗逆性和固氮能力。为了开发一套高效的草木樨转化体系，本研究以白花草木樨为材料，以结瘤基因MaROP6为目的基因，建立了发根农杆菌（Agrobacterium rhizogenes）介导的高效白花草木樨毛状根转化体系。结果表明：利用该方法可以在两周左右获得毛状根，其发生率可达78%，MaROP6基因的毛状根转化率可达70.8%。qRT-PCR鉴定结果表明，MaROP6转基因毛状根的平均相对表达量为对照的36.87倍。毛状根转化体系的建立为白花草木樨基因功能的验证奠定了基础。     

DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: application in experimentally infected chickens

DNA probes specific for Mycoplasma gallisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gallisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. gallisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.     

Pathological changes of tracheal mucosa in chickens infected with infectious laryngotracheitis virus

Six-week-old chickens were inoculated via the posterior thoracic air sac with infectious laryngotracheitis virus. Chickens were sacrificed on various days through day 16 postinoculation (PI), and the trachea was examined by scanning electron microscopy (SEM) and light microscopy (LM). The pathological changes observed on day 1 PI were hypertrophy and hyperplasia of goblet cells. From day 3 PI, the epithelial cells protruded collectively and fused to form syncytia, which contained many intranuclear inclusion bodies. Subsequently, epithelial syncytia desquamated, one after another, and connective tissues were exposed in places. Serofibrinous exudate and detritus were abundant on the surface of the exposed connective tissues and seemed to form a pseudomembrane. On day 5 PI, the remaining epithelial cells began to repair the devastated mucosa just under the pseudomembrane. On day 6 PI, microvillus-rich regenerating epithelial cells were arranged like paving stones. On day 8 PI, the epithelial cells proliferated extensively and formed folds with cyst-like structures. By day 16 PI, the tracheal epithelium was covered with cilia and regained its normal histologic appearance.     

Lectin histochemistry of trachea and lung of healthy turkeys (Meleagris gallopavo) and turkeys with pneumonia

Thirteen lectins were used to characterize lectin-binding specificity of glycoconjugates on sections of formalin-fixed lung and trachea from seven normal turkeys, two turkeys with acute pneumonia, and two turkeys with chronic pneumonia. Neuraminidase was used to digest sialic acid residues. One N-acetylgalactosamine-binding lectin and two N-acetylgalactosamine/galactose-binding lectins stained the apical membrane and cytoplasm of multifocal cells that lined air atria and hyperplastic granular cells. Other lectins in these groups stained ciliated cells of the trachea and bronchi and air capillary epithelial cells. Sialic acid residues were on apical surfaces of ciliated and nonciliated tracheal and bronchial lining cells, air capillary epithelial cells, and vascular endothelial cells. Mannose/glucose-binding lectins stained reticular and elastic fibers in the lamina propria of trachea, primary and secondary bronchi, and the tunica adventitia of arteries and veins. By transmission electron microscopy, colloidal gold-Arachis hypogaea (peanut agglutinin) labeled microvilli on the apical surface of mature granular cells. The L-fucose-binding lectin, in addition to several other lectins, stained nonspecifically in both trachea and lung. These studies show that granular cells that line air atria can be identified with lectins of N-acetylglucosamine and N-acetylgalactosamine/galactose groups, and that apical surfaces of epithelial cells and endothelial cells in the trachea and lung express terminal sialic acid residues.     

Isolation of mycoplasmas from milk, lungs and prepuce of cattle

M. bovis was mainly isolated from 6.25% of milk samples collected from cows with clinical and sub-clinical mastitis. The main mycoplasma strain isolated from the respiratory tract of calves with symptoms of bronchopneumonia was M. bovirhinis (12.1% of samples). M. bovigenitalium was most frequently recovered from bull's prepuce (67.1% of samples).     

Transmission of pathogenic respiratory bacteria to specific pathogen free pigs at slaughter

The purpose of this study was to evaluate the transmission of pathogenic respiratory bacteria to thirteen 5-month-old specific pathogen free (SPF) pigs, during the slaughtering process in a commercial slaughterhouse. Before transportation, the SPF pigs and the lorry were checked to confirm the absence of pathogenic respiratory bacteria.

Nine SPF pigs (group 1) were in contact in a conventional slaughterhouse with finishing pigs, during 4 h before slaughtering. Four SPF pigs (group 2) were slaughtered immediately at arrival in the slaughterhouse.

Five bacterial pathogens (Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis) were detected by PCR, after slaughtering, from nasal cavities, tonsils and trachea in the two groups of pigs. Lung samples were PCR negative. Three and four bacterial species were isolated from the pigs of group 2 and group 1, respectively. Cultures were negative from the lungs.

All the bacterial species present in the SPF pigs were detected by PCR. P. multocida was isolated, from three samples of scalding water before the onset of slaughtering.

Our results suggest that the SPF pigs became contaminated mainly by the slaughterhouse environment and the scalding water. Histological examinations revealed that during scalding, contaminated water could reach the trachea and the lungs of pigs. Checks conducted at slaughter for respiratory disorders have to be carried on, but nasal cavities and tonsils are not appropriate for bacteriological investigations. Moreover, bacteriological results obtained from the lungs of slaughtered pigs have to be used with carefulness.     

An ultrastructural study of the equine lower respiratory tract

The surface features of the lower respiratory tract of 20 clinically normal horses of different ages and types were studied with scanning electron microscopy. Parallel light microscopical and transmission electron microscopical studies were also carried out. The ciliary carpet was virtually complete from the trachea to the lobar bronchi. In small bronchi, ciliation was less complete allowing numerous non-ciliated mucous cells to become obvious. The terminal bronchioles, populated mainly by non-ciliated bronchiolar epithelial cells, had an abrupt junction with alveolar ducts. Interalveolar pores were common particularly in older horses.     

7种优良观赏草光合生理日变化及光响应特征研究

采用便携式LI-6400光合测定仪,选择晴朗天气对观赏草劲芒、柳枝稷、花叶虉草、歌舞芒、矮蒲苇、遍生芒、晨光芒的叶片细胞间CO₂浓度(Ci)、气孔导度(Cond)、气孔限制值、净光合速率(Pn)、瞬时光合有效辐射(PAR)和蒸腾速率(Tr)等有关参数进行测定,并计算各材料的光合饱和点(LSP)和光合补偿点(LCP),研究材料光合日变化及其耐荫性,测定结果表明,7种观赏草中,柳枝稷的光补偿点、净光合速率和水分利用率(WUE)最高,抗旱耐荫。晨光芒和花叶虉草均有较高气孔导度,两者在净光合速率和蒸腾速率方面,晨光芒最高,花叶虉草最低,花叶虉草WUE最低,抗旱性差,在潮湿或水环境中表现好,为具有较强耐荫能力C₃型植物,出现“光合午休”现象,主要由气孔导度下降引起。根据测定的数据及田间不同相对光强下材料的生长情况综合评估,7份材料的耐荫程度由强到弱依次是矮蒲苇、花叶虉草、歌舞芒、遍生芒、劲芒、柳枝稷、晨光芒。     

Evaluation of cytopathologic changes induced in chicken tracheal epithelium by Mycoplasma gallisepticum in vivo and in vitro

Changes in tracheal epithelial surfaces induced by Mycoplasma infection in vivo and in vitro included release of mucous granules followed by exfoliation of ciliated and nonciliated epithelial cells. Light microscopy, scanning electron microscopy, and transmission electron microscopy confirmed that the loss of cilia from individual cells was infrequent. Epithelial cells typically lost their intercellular connections, rounded up, exfoliated, and then lysed--giving rise to a population of cellular organelles, such as mitochondria and cilia intermixed with mucus to form the exudate found within the tracheal lumen. Repair of the epithelial surface was effected by basilar epithelial cells differentiating and filling in the spaces formed by exfoliated cells. These cells were hypertrophied, nonciliated at 14 days after infection in vivo, and covered with microvilli. In sectioned material obtained during the infection, there was increasing epithelial thickness due to cellular infiltration and edema. Tracheal rings in vitro showed similar changes to those seen in vivo, except that exfoliation was more severe and occurred earlier. In addition, there were no cellular infiltration due to the lack of a vascular supply and only a small amount of mucus due to the smaller number of mucous cells available to release into the tracheal lumen.