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)     

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.     

Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant

Objective To investigate the protective efficacy of a pool of denatured membrane protein antigens of Mycoplasma hyopneumoniae (J strain) in the molecular size range 70 to 85 kDa (F3 antigen) in combination with adjuvants for pigs challenged with M hyopneumoniae .
Design A vaccine efficacy experiment with assessment of serum and respiratory tract antibody responses.
Procedure F3 antigens were emulsified with five different adjuvants. To groups of three pigs per vaccine, four vaccines were given by intramuscular injection, and two vaccines, including one of those given intramuscularly, were given by intraperitoneal injection.
Results Compared to six unvaccinated pigs, animals vaccinated with F3 antigen displayed significantly reduced pneumonia (54% reduction in mean lung score) following experimental challenge. Analysis of post-vaccination, pre-challenge IgG and IgA ELISA antibody absorbances in serum and respiratory tract washings revealed no correlation with lung score. Six weeks after challenge, pigs previously vaccinated intramuscularly mostly demonstrated greater IgG and IgA responses in respiratory tract washings, and greater IgG serum antibody responses, than those vaccinated by intraperitoneal injection.
Conclusion Pigs vaccinated with M hyopneumoniae antigens in the molecular size range of 70 to 85 kDa showed a significant reduction in lung lesions compared with unvaccinated control animals after experimental challenge. IgG and IgA antibody concentrations in serum and respiratory tract washings after vaccination do not provide a useful prognostic indicator of protection from enzootic pneumonia.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

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.     

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.     

Histochemical and morphologic changes of porcine airway epithelial cells in response to infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae causes pneumonia in pigs. The effect of infection by this organism on histochemical characteristics of airway mucin within epithelial cells was studied. Seven- to 10-week-old pigs were inoculated intratracheally with M hyopneumoniae or culture broth, and lung tissues were collected from inoculated and control pigs at 2, 4, and 6 weeks after inoculation. Tissue sections were stained with periodic acid-Schiff/Alcian blue, pH 2.5 or high iron diamine/Alcian blue. Histologic features of randomly selected bronchi, bronchioles, and submucosal glands were compared in sections stained with periodic acid-Schiff/Alcian blue. Bronchial goblet cell sulfomucin and sialomucin were quantitated by image analysis of sections stained with high iron diamine/Alcian blue. Bronchi and bronchioles of infected pigs contained proportionately fewer goblet cells with mucin at all stages of infection than age-matched control pigs. Goblet cells in bronchi of infected pigs contained significantly less total mucin and sialomucin, and significantly more sulfomucin than goblet cells of control pigs. Increased sulfated mucin in bronchial goblet cells may reflect altered glycoprotein production or secretion in response to infection with M hyopneumoniae.     

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.     

Effect of Mycoplasma hyopneumoniae colonization at weaning on disease severity in growing pigs

To determine whether Mycoplasma hyopneumoniae colonization at weaning in off-site weaning systems is associated with the severity of respiratory disease due to this agent in growing pigs, we studied 20 groups, each group representing a different week in production, in sow herds at 3 farms of 3000 sows each that had a prevalence of M. hyopneumoniae colonization at weaning higher than 5%. The calculated sample size for assessment at weaning was 39 piglets for each group under study; 39 litters were randomly selected, and 1 piglet was randomly selected from each litter for testing and ear-tagged. In total, 780 piglets were tested. The presence of M. hyopneumoniae in nasal swabs at weaning was established by nested polymerase chain reaction (PCR). All groups were followed until slaughter, at which time blood samples were collected from each ear-tagged pig to test for M. hyopneumoniae antibodies, bronchial swabs were collected for detection of M. hyopneumoniae DNA by nested PCR, and the lung lesion score and percentage of affected lungs in the same animals were calculated. Correlation analyses showed a positive correlation between colonization at weaning and all 4 dependent variables indicating infection at slaughter: average lung lesion score, percentage of affected lungs, presence of M. hyopneumoniae on the bronchial epithelium, and seroconversion. This study provides evidence that severity of the disease can be predicted by the prevalence at weaning in segregated systems. Therefore, strategies focused on reducing colonization at weaning seem to be important elements in the global control of M. hyopneumoniae in segregated production systems.     

Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum

An enzyme-linked immunosorbent assay (ELISA) was evaluated for detection of antibodies (Ab) against Mycoplasma hyopneumoniae and M. flocculare in sera from swine experimentally infected with these agents. In addition, the ELISA was compared with the complement fixation test (CFT), and radial immunodiffusion enzyme assay (RIDEA) for the demonstration of Ab against M. hyopneumoniae. Twenty two 6-week-old swine from a respiratory disease-free herd were divided into five groups. Two or three pigs from each of the four groups were inoculated, respectively, with M. hyopneumoniae or with M. flocculare while two pigs in each group were contact exposed to the inoculated penmates. A fifth group, consisting of three pigs, served as inoculated controls. Pigs inoculated with M. hyopneumoniae began coughing 13 days post inoculation (PI). Antibodies were first detected 2 weeks PI with the CFT, 3 weeks PI with the ELISA, and 5 weeks PI with the RIDEA. With the ELISA and RIDEA, Ab were still detectable one year PI at a very low level. With the CFT, Ab were not detectable in sera from any swine beyond 5 months PI. At necropsy 1 year PI, no lesions were detected in lungs of any of the animals nor were mycoplasmas detected. M. flocculare inoculated or contact-exposed pigs never evidenced clinical signs. Antibodies against M. flocculare were first detected 5 to 12 weeks PI with CFT, and 6 to 12 weeks PI with the ELISA. Peak optical density (OD) values obtained in the ELISA with M. flocculare Ab were as high as the values obtained with peak M. hyopneumoniae Ab titers. Levels of Ab against M. flocculare were at relatively higher OD at 1 year PI than Ab against M. hyopneumoniae. Sera with high levels of Ab against M. flocculare cross-reacted slightly with M. hyopneumoniae antigen in immunoblotting and ELISA.     

Varying effects of infections with Mycoplasma hyopneumoniae on the weight gain recorded in three different multisource fattening pig herds

Pigs in three specialized fattening herds were studied with respect to the effect of infection with Mycoplasma hyopneumoniae on weight gain. Individual pigs were weighed four times at 4-week intervals during the fattening period and their daily weight gain over the rearing period was calculated. A blood sample was collected on each weighing occasion and analysed for the presence of antibodies to M. hyopneumoniae. The lungs of the principals were inspected at slaughter and the extent of pneumonic lesions was registered by a specially developed technique that has been proven to warrant a high degree of repeatability. No serum antibodies to M. hyopneumoniae were detected in one of the herds, and no pneumonic lesions were recorded at slaughter in that herd. In the other two herds, the prevalence of pigs with serum antibodies to M. hyopneumoniae increased from 6 to 54% and from 31 to 81%, respectively, during the fattening period. The prevalence of pneumonic lesions at slaughter in these herds was higher the later the pigs seroconverted. On the other hand, the extension of the lung lesions tended to be higher among pigs that seroconverted early during the rearing period. Infections with M. hyopneumoniae acquired early during the rearing, presumably strengthened by secondary infections and environmental errors, was found to decrease the daily weight gain of the pigs. However, even non-complicated M. hyopneumoniae infections acquired late in the fattening period were associated with reduced daily weight gain. That growth reduction was estimated to be at least 60 g (about 6%) after adjusting for herd, pen, initial weight and sex.     

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.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.     

Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2

The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.     

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.     

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.     

Isolation of Mycoplasma hyopneumoniae from different sampling sites in experimentally infected and contact SPF piglets

The purpose of this study was to determine the optimal route of infection and the optimal sampling sites for the recovery of M. hyopneumoniae, the etiological agent of enzootic porcine pneumonia. Virulence of two strains, BQ 14 and 116, isolated in France in 1975 and 2003, respectively, was also compared. Groups of specific pathogen free piglets were experimentally infected by the intratracheal or intranasal route. One non-inoculated pig was placed in each group of infected pigs to study direct transmission. Two groups were kept uninfected. Coughing was recorded daily. Blood samples, nasal, tonsillar and tracheal swabs and tracheobronchiolar washings were collected weekly. Pigs were killed 27-37 days post-infection. Lung lesions were scored and swabs were collected from nasal cavities, tonsils, trachea, lung, liver and spleen. All the samples, collected from live and dead pigs, were cultured for M. hyopneumoniae recovery. Results showed that both experimentally infected pigs and contact pigs developed enzootic pneumonia, whatever the route of infection and the strain tested. Direct contact transmission occurred quickly. No difference between the two routes of infection or between the two strains tested was evidenced, but high individual variations were observed between pigs. Tracheal swabs and tracheobronchiolar washings were the most effective samples to detect M. hyopneumoniae compared to nasal or tonsillar swabs. Our results also suggested that tracheobronchiolar washings could have an influence on the lesion extent observed at necropsy. M. hyopneumoniae could be re-isolated from liver and spleen of experimentally infected pigs and contact pigs.     

Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs

The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.     

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.