José B. Poveda

Correlating the immune response with the clinical–pathological course of persistent mastitis experimentally induced by Mycoplasma agalactiae in dairy goats

To correlate the clinical course of mycoplasma mastitis with its immune response, right mammary glands of 15 lactating goats were inoculating with 10(10) colony-forming units (cfu) of Mycoplasma agalactiae (Ma). Before sacrificing the animals at 5, 15 or 45 days post-inoculation (dpi), blood Ma antibody titres and milk mycoplasma colony and somatic cell counts were monitored. Ma colonised the mammary gland and milk counts increased to over 10(12)cfu/ml within 5 dpi. During this period, an innate immune response involving neutrophils and macrophages was observed, and Ma antigen appeared in the degenerated acinar epithelium. From 7 dpi, a specific antibody response coincided with reduced viable mycoplasmas in milk. The humoral immune response was limited; by 37 dpi, all animals scored negative for anti-Ma antibodies, and around 10(8)cfu/ml were shed. Results indicate an early immune response to Ma inoculation unable to control mycoplasmal invasion. An ensuing humoral response, despite reducing the mycoplasma burden, leads to chronic, persistent infection.

Quorum-sensing regulator sdiA and marA overexpression is involved in in vitro-selected multidrug resistance of Escherichia coli

OBJECTIVES The role of sdiA in the acquisition of low-level multidrug resistance (MDR) was analysed and compared with that of marA and soxS in two Escherichia coli clinical isolates and two in vitro-selected mutants. METHODS The mutants were developed by growth in lomefloxacin and ceftazidime. The sdiA, marA, soxS, ftsI, tolC and acrB gene transcript levels were determined by RT-PCR. Analyses of 2,4-dinitrophenol susceptibility, the effect of an active efflux inhibitor on antibiotic and mitomycin C susceptibility, beta-lactamase hydrolytic activity, outer and inner membrane proteins and acrR gene sequencing were also performed. RESULTS Both mutants showed elevated marA and sdiA gene transcript levels, which were associated with increased susceptibility to 2,4-dinitrophenol; soxS overexpression was only seen in the mutant selected with ceftazidime. The two mutants showed MDR phenotypes in which ceftazidime, cefpirome and aztreonam MICs increased 4- to 128-fold, in addition to decreased susceptibility to quinolones, chloramphenicol and mitomycin C. The highest ceftazidime MIC in one of the mutants coincided with a frameshift mutation in acrR and the highest transcript level of ftsI (penicillin-binding protein 3), but not with a higher beta-lactamase activity. Likewise, active efflux associated with increased levels of acrB and tolC and decreased OmpF expression contributed to low-level MDR in both mutants. CONCLUSIONS marA and sdiA overexpression was a common feature of multidrug-resistant mutants selected by growth in lomefloxacin and ceftazidime. To our knowledge, this report is the first to describe in vitro selection with a fluoroquinolone or ceftazidime triggering sdiA overexpression in E. coli isolates.

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.

Latent infection of male goats with Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri at an artificial insemination centre.

Contagious agalactia affects goats and is caused by several species of mycoplasma including Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc). Male goats, latently infected with M. agalactiae and Mmc, were identified at a dairy goat breeding artificial insemination centre. In three samplings, conducted over 1 year, ear swabs were assessed for both of the above organisms using culture and PCR techniques. Serological examination for antibodies against these organisms was performed at each time-point and conjunctival, nasal, rectal and preputial swabs were taken from a sub-sample of animals. Mycoplasma mycoides subsp. capri and M. agalactiae were detected in 80 and four ear swabs, respectively and serology confirmed the presence of both agents. A point prevalence of 0.06 goats infected with Mmc at the first sampling point increased to 0.97 at the last sampling, suggesting spread of infection. Both organisms were also detected in preputial and conjunctival swabs suggesting the shedding of these pathogens by other routes. These findings should inform World Organisation for Animal Health (OIE) guidelines on avoiding the introduction of such pathogens into artificial insemination centres and suggest the need to review current recommendations.

Chronological and immunohistochemical characterization of the mammary immunoinflammatory response in experimental caprine contagious agalactia.

To explore the pathogenesis of caprine contagious agalactia (CA), we assessed the ability of Mycoplasma agalactiae (Ma) to modulate the immune system in host tissues by immunohistochemically and chronologically characterizing the main cell subsets present during the mammary immunoinflammatory response. Fifteen lactating goats were inoculated with 10(10) colony-forming units (cfu) of Ma and killed 5, 15 or 45 days post-inoculation (dpi). Blood was taken before necropsy to determine antibodies and milk to determine mycoplasma number. Cells in mammary tissue expressing lysozyme, myeloid-histiocyte antigen (Mac387), major histocompatibility complex class II antigen, immunoglobulin G (IgG), IgA, and CD3, CD4 and CD8 lymphocytes were determined by immunohistochemistry. Results indicate an innate immune response in animals sacrificed at 5dpi, characterized by an abundance of Mac387+ and lysozyme+ cells, that was unable to block or control Ma infection. Elevated numbers of all the cell subsets of the specific immune response (MHC-II+, IgG+, IgA+, CD3+, CD4+ and CD8+ cells) were observed during the subacute stage of the inflammatory process, represented by the 15dpi group. However, these findings could not be correlated with an intense antibody response in blood. The chronic stage of the inflammatory process observed in the goats killed at 45dpi was mainly characterized by expansion of the CD8 compartment at the expense of the CD4 subset leading to a reduced CD4/CD8 ratio. These results contribute to establishing the basic morphological and immunohistochemical characterization of the local immune response against Ma in the goats mammary gland.

The Occurrence of Mycoplasmas in the Lungs of Swine in Gran Canaria (Spain)

The study was conducted to investigate the mycoplasmal flora in the lungs of pigs with enzootic pneumonia at Gran Canaria (Spain). From 54 pneumonic lungs collected at an abattoir, 85 isolates were cultivated. On the basis of cultural and biochemical characteristics, the isolates were preliminarily identified as Mycoplasma species. Using different species-specific PCRs, 40, 27, 11 and 7 of the isolates were identified as M. hyorhinis, M. hyopneumoniae, M. hyosynoviae and M. flocculare, respectively. Nine of the M. hyopneumoniae cultures were found to be in mixed culture with M. flocculare as demonstrated by PCR. By use of a M. flocculare antiserum it was possible to eliminate M. flocculare from M. hyopneumoniae mixed cultures. This study is the first report on isolation of porcine mycoplasmas at Gran Canaria (Spain).

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.