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.

Evaluation of Mycoplasma hyopneumoniae growth by flow cytometry.

AIMS: In the present study we evaluated the potential application of the flow cytometry (FC) technique to determine the growth rates of Mycoplasma hyopneumoniae in a broth medium. METHODS AND RESULTS: The FC analysis was performed using the fluorochromes Syto 9, propidium iodide (PI) or a combination of both dyes and results were compared with those obtained by colour-changing units (CCU) and pH measurements. While CCU counts ml(-1) were higher than those obtained from the FC technique, a good relation between M. hyopneumoniae growth rates was observed in the different phases of the growth curve (logarithmic, stationary and senescence phases). Labelling with Syto 9 alone was sufficient to differentiate M. hyopneumoniae cells with different amounts of nucleic acids, in the stationary and senescence phase of the M. hyopneumoniae growth curve. PI labelling did not detect cell death in the end phase of M. hyopneumoniae growth. CONCLUSIONS: These data show that FC is a very useful, practical and fast technique to study the growth rates of M. hyopneumoniae in broth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is to our knowledge the first application of FC to the study of M. hyopneumoniae growth in broth culture.

Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain.

A field comparison of the interferon-gamma (IFN-gamma) assay and the single intradermal cervical tuberculin (SICT) test for the diagnosis of bovine tuberculosis was conducted. A total of 1136 cattle belonging to 85 herds placed in 'Castilla y León' (northwestern Spain) were chosen, and 21 of these herds were subjected to the diagnostic assays two or three times at intervals of at least 4 months. All the animals positive to any of the tests were slaughtered and tuberculosis was confirmed by culture isolation method (CIM) and further identification by means of PCR. Only 10.6% of cattle reacted with the bovine PPD in the SICT test, a percentage that increased to 12.8% in the IFN-gamma assay. The sensitivity of the IFN-gamma assay compared to CIM was shown to be higher (84.9%) than that of the SICT test (80.2%), but the combination of both tests offered the highest sensitivity (92.9%). The number of false positive reactors (those animals in which CIM was negative) was considerably higher for the IFN-gamma assay than for the SICT test and, conversely, the number of false negative animals (M. bovis isolation but negative immunological result) was higher for the skin test than for the interferon assay. In the herds tested twice, tuberculosis was eradicated after the second cycle of testing in 50%, and in 75% after the third cycle in herds tested three times. The combination of these two techniques instead of separately seems, therefore, to be useful in eradication programmes against bovine tuberculosis.

Comparison of different methods for diagnosis of bovine tuberculosis from tuberculin- or interferon-gamma-reacting cattle in Spain.

Of 1479 cattle from herds in Northwestern Spain previously diagnosed as tuberculosis (TB) positive, 218 animals which gave a positive tuberculin or interferon-gamma reaction were examined at the slaughterhouse. Medial retropharyngeal and caudal mediastinal lymph nodes, and any tissues containing lesions suspected to be tuberculous, were removed and submitted to the laboratory. Three techniques for diagnosis of TB were used: post mortem examination (PME), smear staining by means of auramine O method (AOM), and culture isolation in Coletsos and Lowenstein-Jensen media followed by confirmation of M. tuberculosis complex organisms using PCR (CIM-PCR). Only 123 (29.9%) of the 412 samples collected showed typical tuberculous lesions. Confirmed M. tuberculosis complex organisms were isolated in 144 cases, 114 of which were from tissues showing lesions (success rate of 92.8%). Smears were found positive in 113 cases, 96 of which came from lesions suspected to be tuberculous (success rate of 78.0%). The sensitivities of CIM-PCR compared with those of PME and AOM were 92.7% and 85.7%, respectively. Statistical analysis revealed that PME and AOM are good indicators of the presence of M. tuberculosis complex organisms in tuberculin- or interferon-gamma reacting cattle.

The insertion element IS 6110 is a useful tool for DNA fingerprinting of Mycobacterium bovis isolates from cattle and goats in Spain.

A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.

Seroepidemiological survey of Q fever in León province, Spain.

A seroepidemiological survey, using an indirect immunofluorescence test against Coxiella burnetii (antigenic phase II), was carried out in León province, north-western Spain. A total of 406 serum samples was collected from people (from 15 to more than 65 years old) living in a rural environment during the winter and spring of 1994. The overall prevalence was 40.6%, titres ranged from 1:80 to 1:640, and a titre of 1:80 was encountered among 60.6% of positive samples. A significant higher prevalence was observed among males globally, as well as among those aged 15-44 and 45-64 years old; however, no difference was encountered among males and females older than 64 years. In the same way, a significant higher Q fever prevalence was observed among individuals having occupations related to agriculture and among those having close relationship with animals.