Hyopneumoniae Isolates Research Articles

Protein and Antigenic Variability among Mycoplasma hyopneumoniae Strains by SDS-PAGE and Immunoblot

Porcine enzootic pneumonia (PEP), with Mycoplasma hyopneumoniae as the primary agent, is a chronic respiratory disease that causes major economic losses to the pig industry worldwide. The aim of this work was to analyse 18 field strains of M. hyopneumoniae isolated in Gran Canaria (Spain) and the reference M. hyopneumoniae strain by SDS-PAGE and immunoblot. A monoclonal antibody (MAb) against the membrane protein p46 reacted with all the strains in this study. In contrast, a purified polyclonal antibody (PAb) against the cytoplasmic protein p36 reacted with this protein in only 10 strains. A MAb against the adhesin protein p97 stained multiple proteins of different sizes and with different intensities. Different antigenic patterns in the same M. hyopneumoniae strains were also observed after different numbers of passages in culture medium. Furthermore, variability in the staining of the 36 kDa protein was observed, depending on whether the p36 PAb or the antiserum against M. hyopneumoniae reference strain was used. It is concluded that local M. hyopneumoniae field isolates in Gran Canaria are characterized by protein diversity.

The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel electrophoresis

Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol is described for the typing of Mycoplasma hyopneumoniae isolates. The potential of this technique was demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as from different herds. The use of two different restriction enzymes, SalI and ApaI, was evaluated. For each enzyme, the resulting restriction profiles were clustered using the unweighted pair group method with arithmetic means (UPGMA). For both obtained dendrograms, the included isolates of the related M. flocculare species clustered separately from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns of the M. hyopneumoniae isolates of different herds were highly diverse and clustered differently in both dendrograms, illustrated by a Pearson's correlation coefficient of only 0.33. A much higher similarity was observed with isolates originating from different pigs of a same herd. The PFGE patterns of these isolates always clustered according to their herd and this for both dendrograms. In conclusion, the results indicate a closer relationship of M. hyopneumoniae isolates within a herd compared to isolates from different herds and this for both restriction enzymes used. Since the described PFGE technique was shown to be highly discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology of M. hyopneumoniae.

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.

In vitro susceptibilities of recent field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to valnemulin (Econor®), tiamulin and enrofloxacin and the in vitro development of resistance to certain antimicrobial agents in Mycoplasma hyopneumoniae

The in vitro activities of valnemulin (Econor®) and two other antimicrobial agents were determined against recent field strains of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae using a broth microdilution method. Valnemulin showed exceptional activity against M hyopneumoniae ( MIC90 0·0005 μg ml −1) and M hyosynoviae ( MIC range 0·0001 μg ml −1 to 0·00025 μg ml −1) field strains. Tiamulin was 100-fold less active ( MIC90 0·05 μg ml −1) and enrofloxacin 20-fold less active ( MIC90 0·01 μg ml −1) than valnemulin against M hyopneumoniae field isolates and 20-fold to 25-fold less active ( MIC range 0·0025 μg ml −1 to 0·005 μg ml −1) and 400-fold to 500-fold less active ( MIC range 0·05 μg ml t1̄ to 0· 1 μg ml −1) respectively against M hyosynoviae field isolates. No significant resistance developed to valnemulin or tiamulin in the type strain of M hyopneumoniae (strain J) or in a recent field isolate ( MEVT G23) exposed to 10 in vitro passages in broths containing these antibiotics. Only slight resistance to oxytetracycline was observed. High resistance to tylosin developed in both M hyopneumoniae strains within five to seven in vitro passages in tylosin-containing broth. Providing that similar results are obtained in vivo under field conditions, valnemulin may well prove to be effective in the treatment of enzootic pneumonia and acute polyarthritis in pigs.

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.