Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes.

BACKGROUND: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. METHODS: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. RESULTS: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10-4 and 10-5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. CONCLUSIONS: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

Relationship Between Expression of Interleukin-5 and Interleukin-13 by Epithelial Cells and Bronchiolar Changes in Pigs Infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mh) is a bacterium that specifically infects the surface of bronchi and bronchioles of pigs without invading the host cells, and it is considered to be the primary agent of porcine enzootic pneumonia (PEN). The present study investigates the morphological and immunohistological changes induced in bronchiolar epithelium by Mh infection. Lungs from 20 pigs with naturally occurring Mh pneumonia were compared with those from 10 uninfected controls. Bronchiolar epithelial height, inflammatory infiltration, hyperplasia of bronchus-associated lymphoid tissue (BALT) and mucin subtype MUC5AC-producing cells significantly increased in all infected animals. Mh antigen was detected in association with the cilia of the bronchial and bronchiolar epithelium. Interleukin (IL)-5 and IL-13 were expressed consistently by epithelial and mononuclear cells of the airways of infected animals. The expression of these cytokines in the bronchial and bronchiolar tissues is related to the histological changes of PEN.

Immunohistochemical labelling of cytokines in calves infected experimentally with Mycoplasma bovis.

To gain further insight into the pathogenesis of Mycoplasma bovis-associated pneumonia, cytokine expression in different pulmonary compartments was examined. The expression of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and interferon (IFN)-γ was examined immunohistochemically in the lung of 10 calves infected experimentally with M. bovis. M. bovis antigen was located in respiratory epithelial cells and within inflammatory cells in the airway lumina. Immunolabelling for TNF-α, IL-4 and IFN-γ was usually associated with inflammation, particularly in macrophages and lymphocytes in hyperplastic bronchus-associated lymphoid tissue (BALT), in thickened alveolar septa and in the bronchoalveolar exudate of infected animals. In M. bovis infection, macrophage and lymphocyte activation results in expression of a number of cytokines capable of inducing lung lesions and hyperplasia of the BALT. The cytokines examined likely play a role in pulmonary defence against M. bovis infection.

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.

Mycoplasma neophronis sp. nov., isolated from the upper respiratory tract of Canarian Egyptian vultures (Neophron percnopterus majorensis).

Six strains with the typical characteristics of mycoplasmas were isolated from the tracheae of six Canarian Egyptian vultures (Neophron percnopterus majorensis). The results of biochemical, serological and molecular genetic studies showed that the isolates were nearly identical and that they could be considered as representing a novel species of the genus Mycoplasma. Colonies possessed the typical fried-egg appearance and electron micrographs revealed a pleomorphic cellular morphology with the lack of a cell wall. The isolates hydrolysed arginine and required sterol for growth but did not ferment glucose or hydrolyse urea. We propose that the isolates be assigned to a novel species,Mycoplasma neophronis sp. nov. The type strain is G.A.(T) ( = DSM 24097(T) = ATCC BAA-2157(T)). The antiserum of strain G.A.(T) has been deposited in the Mollicutes collection at Purdue University (Indiana, USA).

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.

In vitro susceptibilities of field isolates of Mycoplasma agalactiae.

In order to determine how widespread antibiotic resistance has become to standard treatments, the in vitro susceptibilities of 28 Mycoplasma agalactiae Spanish field isolates to 16 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials based on minimum inhibitory concentration (MIC)(90) values were fluoroquinolones, tetracyclines and macrolides. Two strains were tetracycline resistant. Streptomycin, erythromycin and nalidixic acid resistance was observed in all strains.

In vitro susceptibilities of Mycoplasma putrefaciens field isolates.

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.

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. METHODS AND RESULTS: Flow cytometry was able to detect Mycoplasma hyopneumoniae inhibition at 12 h postincubation, whereas the results obtained by the traditional method were only obtained at 48 h, when a visible change in the medium had occurred. At 48 h, both methods gave the same result for eight antibacterial agents, whereas flow cytometry gave slightly higher MIC values for one antibacterial agent (tylosin). This was attributed to the fact that the M. hyopneumoniae growth that had occurred in those tubes was not enough to visibly change the colour of the medium. A good relationship was found between the flow cytometry and the traditional method. CONCLUSION: Flow cytometry was found to be a good method for the determination of antimicrobial MICs in M. hyopneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: The flow cytometric method allows the determination of the response of M. hyopneumoniae to each of the antibacterial agents in near real time, and has potential for the identification and study of resistant subpopulations.

Immunohistochemical detection of interleukin-12 and interferon-gamma in pigs experimentally infected with Mycoplasma hyopneumoniae.

The expression of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) was examined immunohistochemically in the lungs of pigs aged 21 days infected experimentally with Mycoplasma hyopneumoniae (Mh). Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 7 to 35 days post-infection (dpi). Immunolabelling for IL-12 and IFN-gamma was usually associated with inflammation, particularly in macrophages and lymphocytes in the thickened alveolar septa and in the hyperplastic bronchus-associated lymphoid tissue (BALT). Cells positive for both cytokines were detected at 7 dpi, their numbers increasing at 14 and 21 dpi, and slightly decreasing thereafter. The results suggest that IL-12 and IFN-gamma play a role in pulmonary defence mechanisms against Mh infection.

First isolation of Mycoplasma capricolum subsp. capricolum, one of the causal agents of caprine contagious agalactia, on the island of Lanzarote (Spain).

During an unusually long period of bad weather, several outbreaks of caprine contagious agalactia (CCA) were reported in a number of flocks on the island of Lanzarote (Canary Islands, Spain). Clinical and subclinical mastitis in lactating goats and some cases of arthritis and pneumonia in kids were observed in the affected flocks. Mycoplasma capricolum subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with M. mycoides subsp. mycoides "large colony type" (Mmm LC) in two flocks. This is the first report of an isolation of M. capricolum subsp. capricolum on the island of Lanzarote. The finding is of epidemiological importance and could complicate plans to control the disease. The significance of this mycoplasma species in association with CCA must now be studied in detail.

In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials.

In vitro susceptibilities of 16 Mycoplasma mycoides subsp. mycoides large colony type field isolates to 15 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials were fluoroquinolones, tetracyclines and macrolides, with MIC values under 2 microg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.

Sudden death associated with Clostridium sordellii in captive lions (Panthera leo).

In the spring of 2003, a series of sudden deaths in a group of adult lions (Panthera leo) with a previous history of depression, inanition, and lethargy, was investigated. Five animals died within 24 to 36 hours after onset of signs of disease. Serologic screening for viral disease detection was negative, evidence of parasites was not detected, and results of a complete blood count and serum biochemical analysis were within reference intervals in all lions. The most relevant lesions observed were multiple areas of necrosis and hemorrhage in the intestinal outer muscular layer, and cellulitis with an intense bloody edema in the mesenteric and the pericardial fat tissue. On the basis of the fulminant course of the disease, the gross and histologic findings, and the isolation and identification of Clostridium sordellii, a diagnosis of infectious myositis and cellulitis associated with acute clostridiosis was made. To the authors' knowledge, this is the first report of sudden death associated with C. sordellii in felines.

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting.

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium.

AIMS: The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined. METHODS AND RESULTS: The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, - CFUs). There was a good correlation (linear regression, r(2) = 0.93) between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count method (CFU ml(-1)). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10(4) cells ml(-1) and 10(3) CFU ml(-1), respectively. FC method allowed results in 20-30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU). CONCLUSION: Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).

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).

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.

Microbiological survey for Mycoplasma spp. in a contagious agalactia endemic area.

In this work, we report a microbiological survey for Mycoplasma spp. undertaken between 2001 and 2002 in 28 goat herds in Gran Canaria, Spain, an area where contagious agalactia is endemic. All herds were randomly selected and represented approximately 15.5% of the total goat population of the island. A variable number of milk, articular and auricular swab samples were collected from each flock and cultured in specific mycoplasma culture media. There was a total of 38.5% positive flocks from which 37 mycoplasma isolates were obtained. In contrast with previous data obtained in Spain, our results showed that the large colony variant of M. mycoides subsp. mycoides (Mmm LC) was the most commonly isolated agent associated with contagious agalactia. This species was isolated from 90% of the positive herds and accounted for 54.1% of all isolations. M. agalactiae was isolated from 40% of the positive herds (27% of all isolations) and in six herds M. arginini was isolated (18.7% of all isolations). No M. capricolum or M. putrefaciens strains were isolated. Mycoplasmas were isolated from 21 milk samples, 15 ear canals swabs and one articular sample. The association of several species was reported in several herds. These results are at variance with previous serological studies, which indicated a higher disease prevalence, and suggest that it could be necessary to use detection techniques such PCR to confirm the existence of contagious agalactia in goats.

Relationship between rheumatoid arthritis and Mycoplasma pneumoniae: a case-control study.

OBJECTIVE: Rheumatoid arthritis (RA) has a complex and multifactorial aetiology. Infectious agents could start this disease. The majority of the characteristics of this infirmity can be observed in chronic arthritis produced by mycoplasmas in animals. In this study the association between Mycoplasma pneumoniae and RA has been evaluated. METHODS: A case-control study was performed. Sera taken from 78 RA patients and from 156 controls were analysed to ascertain the levels of immunoglobulin G (IgG) against M. pneumoniae. Other variables, like age, gender, work status, history of pneumonia, etc., were recorded in a questionnaire. RESULTS: The presence of antibodies against M. pneumoniae was associated with RA (odds ratio=2.34, P<0.001). CONCLUSIONS: The results suggest that M. pneumoniae could be a cofactor in the pathogenesis of RA; however, more studies need to be done.