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.     

Ultrastructure of the hydrophobic gastric surfactant barrier in the dog

OBJECTIVES: To confirm the hydrophobicity of the luminal surface of the canine stomach and to elucidate the ultrastructure of the lining imparting that property. DESIGN AND PROCEDURES: Gastric and duodenal mucosal samples from eight dogs were collected immediately after euthanasia and subjected to contact angle measurement using a goniometer. Other samples were examined by electron microscopy following a fixation procedure known to preserve phospholipids and oligolamellar structures. RESULTS: Contact angles for the canine gastric mucosal surface (85.1 +/- 5.5) were significantly greater (P < 0.0001) than for the duodenum (24.0 +/- 1.7). Electron microscopy revealed the existence of surfactant as abundant osmiophilic phospholipid material within the gastric and duodenal mucosae. CONCLUSION: We have confirmed the hydrophobic nature of the canine gastric mucosa whereas the luminal surface of the duodenum is hydrophilic. We propose that the water-repellent nature of the canine gastric lining contributes to the 'gastric mucosal barrier' and is imparted by an oligolamellar layer of surface-active phospholipid ('gastric surfactant') adsorbed to the surface. Both gastric and duodenal mucosae may also utilise phospholipids as an intercellular defense mechanism in the event that tight junctions are breached by acid. It is tempting to speculate that a deficiency of gastric phospholipids may predispose dogs to ulcers. Further, exogenous administration of phospholipids may be useful in preventing gastric ulceration.     

Hemagglutination and hemagglutination inhibition of turkey red blood cells with Mycoplasma hyopneumoniae

The ability of Mycoplasma hyopneumoniae to agglutinate RBC was evaluated to develop an in vitro cytadsorption assay. Using swine RBC in a microtitration hemagglutination test, no agglutination or partial agglutination was detected. Comparison of RBC from various other species indicated that improved hemagglutination was obtained with RBC from turkeys. This hemagglutination was detected only when mycoplasma cells used in the assay had been frozen and thawed, heated at 50 C for 30 minutes, or treated with trypsin. Treatment of RBC with trypsin or neuraminidase enhanced hemagglutination. Possible surface lectin activity in M hyopneumoniae was evaluated by use of carbohydrates in a blocking assay; hemagglutination was not inhibited by any of 13 carbohydrates evaluated. Mycoplasma hyopneumoniae convalescent porcine serum and monoclonal antibodies against 2 M hyopneumoniae immunogens of molecular weights of 64,000 and 41,000 inhibited hemagglutination.     

Evidence for surfactant contributing to the gastric mucosal barrier of the horse

This study was undertaken to determine the hydrophobicity of the luminal surface of the equine stomach and to elucidate the ultrastructure of the lining imparting that property. Gastric and duodenal mucosal samples from 5 horses were collected immediately after euthanasia and subjected to surface contact angle measurement using a goniometer. Gastric mucosal samples from 4 horses and a foal were examined by electron microscopy following a fixation procedure known to preserve phospholipids and oligolamellar structures. Contact angles for the equine gastric glandular mucosal surface (mean +/- s.e. 78.0 +/- 11.0 degrees) were greater than for the duodenum (33.4 +/- 8.7 degrees), (P = 0.003). The contact angles for gastric squamous tissue (50.4 +/- 4.5 degrees) tended to be greater than for duodenum (P = 0.15). Electron microscopy revealed the existence of surfactant as abundant osmiophilic phospholipid material within both squamous and glandular gastric mucosae. These results indicate the hydrophobic nature of the equine gastric mucosae. We propose that the water-repellent nature of the stomach contributes to the 'gastric mucosal barrier' and is imparted by surface-active phospholipid adsorbed to the surface. Phospholipids may also be utilised as a physical barrier to back-diffusion of acid by lining intracellular canaliculi and oxyntic ducts where other defence mechanisms are absent.     

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.     

Comparison of hydrophobic properties of thoracic duct lymph chylomicrons from rats given different fats or oils by gavage

Lipoprotein aggregation is generated by hydrophobic nature of lipoproteins that is known to be one of the causes of atherosclerosis. Low density lipoproteins (LDL) has been extensively studied in this respect but not chylomicrons. There is strong evidence that post‐prandial triacylglycerol‐rich lipoproteins are atherogenic. Because biophysical properties of lipoproteins are largely determined by their lipid compositions, hydrophobic nature of thoracic lymph duct chylomicrons obtained from rats given different fats or oils by gavage was investigated by vortexing‐induced aggregation and hydrophobic interaction chromatography. Contrary to LDL, vortexing did not cause aggregation in chylomicrons. Vortexing of fish oil and butter chylomicrons resulted in more prominent reduction in absorbances compared with chylomicrons from other sources that might indicate less micelle stability. Hydrophobic interaction chromatography of fish oil, palm oil and olive oil chylomicrons yielded three fractions, whereas that of sunflower, margarine and butter chylomicrons gave rise to two fractions. These results suggest that surface hydrophobicity of chylomicrons might be heterogenous. Our results also demonstrate that fish oil chylomicrons have less hydrophobicity and lower stability against vortexing compared with chylomicrons from other sources. Considering beneficial effects of fish oil in cardiovascular health, less hydrophobicity together with lower stability might provide an additional atherogeneicity index for lipoproteins.     

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.     

Comparison of surface hydrophobicity of piliated and non-piliated clones of Corynebacterium renale and Corynebacterium pilosum

Piliated (P+) and non-piliated (P-) clones of Corynebacterium renale and C. pilosum were similar in hydrophobicity as measured by hydrophobic interaction chromatography, bacterial adherence to hydrocarbons and the salt aggregation test. Therefore, the previously reported adherence of P+ clone to various cells, which is more effective than that of P- clone, may be uncorrelated with the degree of hydrophobicity of both clones of these bacteria. Hydrophobicity of P+ and P- clones was found to be high when measured by hydrophobic interaction chromatography and bacterial adherence to hydrocarbons, but low when measured by the salt aggregation test.     

Mycoplasma hyopneumoniae increases the susceptibility of pigs to experimental Pasteurella multocida pneumonia.

The interaction between Mycoplasma hyopneumoniae and Pasteurella multocida in experimental pneumonia was investigated in conventional pigs. The experimental animals were 49 days old when inoculated with M. hyopneumoniae; they were inoculated with P. multocida after 23 days, and killed 13 days later. In pigs inoculated only with P. multocida, clinical signs and lung lesions were not observed, and the agent was not recovered. Pigs inoculated with M. hyopneumoniae developed fever, moderate cough and dyspnea which tended to disappear, and small proliferative lung lesions from which M. hyopneumoniae was isolated. Pigs inoculated with both agents had higher fever, severe cough and dyspnea which tended to aggravate, and extensive exudative lung lesions from which organisms were isolated. All animals had similar growth rates, but the group infected with both agents consumed 60% more food. Therefore, M. hyopneumoniae causes mild pneumonia, whereas P. multocida is not pathogenic alone but aggravates the pneumonia initiated by M. hyopneumoniae.     

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.     

Identification of Mycoplasma hyopneumoniae in formalin-fixed porcine lung, using an indirect immunoperoxidase method

An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.     

Assessment of antibody response of swine infected with Mycoplasma hyopneumoniae by immunoblotting

An immunoblot procedure was used to evaluate porcine antibody response to inoculation with Mycoplasma hyopneumoniae. Mycoplasmas solubilized with sodium dodecyl sulfate were used as antigens. Antibodies to 5 antigens, estimated to be of molecular weight (mol wt) 110,000, 64,000, 50,000, 41,000, and 36,000, were detected in sera collected during the course of induced mycoplasmal pneumonia. Mycoplasma hyopneumoniae antigens, mol wt 110,000, 50,000, 41,000, and 36,000, cross-reacted with M flocculare when antigen prepared from M flocculare or hyperimmune serum against it were used in the immunoblot procedure. The 36,000-dalton (D) antigen reacted with M hyopneumoniae and M hyorhinis convalescent sera. The 64,000-D M hyopneumoniae antigen was the only antigen that did not cross-react with M flocculare or M hyorhinis. Exposure of immunoblot strips with antigens to trypsin before reacting them with the convalescent sera abolished binding ability of the 110,000-D and 36,000-D antigens, but had no effect on binding by 64,000-D, 50,000-D, or 41,000-D antigens. None of the 5 antigens bound to 11 lectins.     

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.     

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.     

Treatment of experimental enzootic pneumonia of the pig by norfloxacin or its 6-chloro analogue

The 6-chloro analogue of norfloxacin (compound A) administered continuously in the feed at 400 ppm for 21 days markedly reduced the extent and activity of pneumonic lesions in pigs with pneumonia induced experimentally with an homogenate of pneumonic lung and broth cultures of Mycoplasma hyopneumoniae. Norfloxacin at 100 ppm or compound A at 200 ppm in the feed did not reduce the extent of lung lesions, although half the pigs treated with norfloxacin had lesions which appeared histologically to be healing. M hyopneumoniae was detected either by culture or immunofluorescence in the lungs of 60 per cent of the pigs treated with compound A at 400 ppm compared with all the pigs in the other groups. These results were related to the amount of drug in the lungs and body fluids during therapy. Only compound A at 400 ppm produced concentrations in the lungs and bronchial secretions exceeding the minimum inhibitory concentration against M hyopneumoniae. Mycoplasmacidal concentrations were not reached either in the lungs or bronchial secretions which might account partly for the frequent detection of M hyopneumoniae in the lungs after treatment. Drug resistance did not appear to be responsible for the persistence of M hyopneumoniae in vivo since the M hyopneumoniae isolates from the pigs after therapy were sensitive in vitro to both quinolones. As daily weight gain and feed-conversion efficiency improved in all groups of treated pigs compared with the controls, these effects were probably unrelated to the antimycoplasmal activities of the two quinolones.     

Surface hydrophobicity of Staphylococcus intermedius and Staphylococcus hyicus.

Surface hydrophobicity of 90 Staphylococcus intermedius and 55 S hyicus isolates was evaluated using the hexadecane adherence assay and the ammonium sulphate salt aggregation test. A strongly positive hydrocarbon adherence in the hexadecane adherence assay was demonstrated in 11 per cent of the S intermedius isolates and 7 per cent of the S hyicus isolates. Bacterial aggregation in 1.6 M, or less, ammonium sulphate was observed in 28 per cent of the S intermedius isolates and 37 per cent of the S hyicus isolates. There was no statistical correlation between the two assays. The adherence of both bacterial species to hexadecane was eliminated when the cells were first treated with pronase and trypsin, while it was mildly enhanced by prior heat treatment (60 degrees C and 95 degrees C for up to three hours). In contrast, aggregation of S intermedius in ammonium sulphate was not influenced by trypsin pretreatment, and aggregation of both bacterial species was diminished, or eliminated, with pronase or prior 95 degrees C heat treatment. Surface hydrophobicity, as measured in both assays, appeared to have no relationship with growth patterns in serum soft agar or production of slime. Similarly, the presence or absence of substantial surface receptor activity to fibrinogen, fibronectin or IgG did not appear to be related to surface hydrophobicity.     

Isolation and trypsin-enhanced propagation of turkey enteric (bluecomb) coronaviruses in a continuous human rectal adenocarcinoma cell line

Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.     

Difference in hydrophobicity of human and animal IgA.

We have used hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B to compare the hydrophobicity of human IgA with the IgAs of eight animal species. The results suggest that the degree of difference between the various IgAs increases with increasing phylogenetic distance.     

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.     

Evaluation of tiamulin for treatment of mycoplasmal pneumonia in swine

During 3 trials, using affected pigs of various ages, tiamulin was evaluated for treatment of experimentally induced mycoplasmal pneumonia. Pneumonia was induced in respiratory tract disease-free swine by intratracheal inoculation of a lung homogenate containing Mycoplasma hyopneumoniae. Eleven days after inoculation, when more than 20% of pigs were coughing, pigs were allotted to 3 or 4 groups (n = 8 pigs each) and were given regimens of no medication or 60 mg, 120 mg, or 180 mg of tiamulin/L of drinking water for 10 days. Twenty-one days after cessation of medication, pigs were euthanatized and then were necropsied. Results obtained from the 3 trials did not indicate significant difference among treatment groups in severity of macroscopic or microscopic lesions induced by M hyopneumoniae or in detection of M hyopneumoniae by use of immunofluorescent technique. Clinical evaluations, daily gain, and feed efficiency did not differ significantly among treatment groups. In this study, tiamulin administration did not have beneficial effects in swine with mycoplasmal pneumonia.