P. Assunção

Serological study of contagious agalactia in herds of goats in the Canary Islands

An indirect ELISA, using local strains of Mycoplasma agalactiae and Mycoplasma mycoides subspecies mycoides large colony (MmmLc), was applied to evaluate the seroprevalence of M agalactiae and MmmLc in flocks of goats on each of the Canary Islands. In total 3890 samples of serum were collected from 204 flocks. The results indicated that the seroprevalence of both organisms is high on all the islands; average values of 55 per cent and 67 per cent were recorded, respectively, for M agalactiae and MmmLc.

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.

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae.

Aim:  In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MICs) of nine antibacterial agents (enrofloxacin, ciprofloxacin, oxytetracycline, chloramphenicol, tylosin, lincomycin, gentamycin, spectinomycin and streptomycin) against M. hyopneumoniae.

Flow cytometric determination of the effects of antibacterial agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides large colony type.

ABSTRACT Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.

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.

Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate.

Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae

In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12‐h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real‐time at the single‐cell level.

Detection of mycoplasmas in goat milk by flow cytometry

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large‐colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 103–104 cells ml−1. Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20–30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples.

In Vitro Susceptibilities of Mycoplasma putrefaciens Field Isolates

ABSTRACT MICs were determined for 15 antimicrobial agents against 37 Mycoplasma putrefaciens isolates. The most effective antimicrobial drug classes were the fluoroquinolones, the tetracyclines, the lincosamide lincomycin, and the macrolides. The susceptibility profile of the isolates correlated with the geographic origin. This is the first report of decreased susceptibility to the macrolides, lincomycin, and the tetracyclines in M. putrefaciens strains.

In vitro activity of tylvalosin against Spanish field strains of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is involved in the porcine enzootic pneumonia and respiratory disease complex; therefore, the search for new treatment options that contribute to the control of this organism is relevant. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of tylvalosin and 19 other antimicrobial agents against 20 Spanish field isolates of M. hyopneumoniae were determined using the broth microdilution method, with the type strain (J) as a control strain. Tylvalosin had MIC50 and MIC90 values of 0.016 and 0.06 µg/ml, respectively, and was the second-most effective of the assayed antibiotics, after valnemulin. Tiamulin, tylosin and lincomycin were also among the antibiotics with the lowest MIC50 and MIC90 values against the 20 field isolates (0.06–0.25 µg/ml). However, resistance to tylosin and spiramycin, which like tylvalosin, are 16-membered macrolides, was observed. The MIC50 and MIC90 values for ciprofloxacin and enrofloxacin ranged from 0.125 to 1 µg/ml; the corresponding values ranged from 2 to 4 µg/ml for oxytetracyline, which was the most active tetracycline. Furthermore, tylvalosin and valnemulin exhibited the highest bactericidal activities. In conclusion, the macrolide tylvalosin and the pleuromutilin valnemulin exhibited the highest in vitro antimicrobial activities against M. hyopneumoniae field isolates in comparison with the other tested antibiotics.