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.

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 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.

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.

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.

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.

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.

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.

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.

An outbreak of encephalitis in pigeons (Columba livia) in the Canary Islands (Spain).

An encephalitic disease in pigeons, characterized by paralysis of extremities, torticollis, head tremor, and diarrhea, is described. The negative titers in the hemagglutination tests, the presence of Feulgen-positive intranuclear inclusions in the cells of the granular layer of the cerebellum and the fibroblast culture, and the presence of viral particles with an icosahedral-herpetic morphology and a diameter of 100-170 nm all indicate that the etiology of this outbreak may be related to the encephalitis caused by herpesvirus, at present diagnosed only in Iraq and some African countries.

Immunohistochemical characterization of lung lesions induced experimentally by Mycoplasma agalactiae and Mycoplasma bovis in goats.

Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. Both Mycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3(+)T lymphocyte, and the ratio of CD4(+): CD8(+)cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4(+)T lymphocytes, plays a prominent role in the acute and subacute phase of these infections.

Isolation of Mycoplasma mycoides, mycoides (LC variant), from two naturally aborted caprine fetuses.

Described in this study are 2 cases of caprine abortion associated with the LC type of Mycoplasma mycoides , subsp. mycoides . This mycoplasma and Mycoplasma mycoides , subsp. capri had been previously reported in adult goats in this herd. The abortion took place in the latter part of gestation. Samples from heart blood, lung and pleural exudate were collected for the isolation of mycoplasmas and other bacterials in both fetuses. Two strains of Mycoplasma mycoides , subsp. mycoides (LC variant) were isolated. The only gross lesion of the internal organs in the aborted fetuses was congestion of the lungs. Microscopic lesions were encountered in the lungs, and these were characterized by patchy to diffuse pneumonia. Exfoliated cells, many alveolar macrophages, scattered neutrophils and lymphocytes were seen in the lumen of the terminal airways and alveolar spaces. This report appears to be the first isolation of Mycoplasma mycoides , subsp. mycoides (LC variant) from aborted caprine fetuses.

A pathological and immunohistochemical study of caprine pleuropneumonia induced by subspecies of Mycoplasma mycoides.

In a population of 700 goats, 150 died; of these, 29 were necropsied. Ten of the 29 goats had pleuropneumonia. Mycoplasma mycoides subsp. mycoides (Large Colony) (MmmLC), Mycoplasma mycoides subsp. capri (Mmc), and Pasteurella multocida were isolated from five of the pleuropneumonic goats. Gross and microscopical lesions were typical of caprine pleuropneumonia (CPP), with bronchopneumonia, fibrinopurulent or fibrinonecrotic pleuropneumonia and dilatation of the interlobular septa and pleura. Immunohistochemical examination with antisera against MmmLC and Mmc showed mycoplasma antigens in all 10 goats with CPP. In all cases, both MmmLC and Mmc antigens were detected together. Mycoplasma antigens were present in the lumina of the airways and alveoli, mainly inside the cytoplasm of neutrophils and macrophages, but extracellular antigen was demonstrated in areas of necrosis. Pasteurella antigens were detected in four of the 10 animals with CPP. From the histological, immunohistochemical and microbiological results it was concluded that the two mycoplasmas, acting together, caused the pleuropneumonia, with P. multocida playing a subsidiary synergistic role.

Association of cilia-associated respiratory (CAR) bacillus with natural chronic tracheitis in goats.

A histological, histochemical and immunohistological study of the respiratory tract of 83 slaughtered goats (50 adults and 33 kids) is described. Cilia-associated respiratory (CAR) bacillus was detected by means of the Warthin Starry method in the tracheal epithelium of seven (21.2%) of the kids and 16 (32%) of the adult goats. A chronic diffuse tracheitis characterized by mixed lymphocyte and plasma-cell infiltration was found in all seven kids and in 17 adults, including the 16 infected with the CAR bacillus. Although not proved, it is possible that the CAR bacillus caused the chronic tracheitis. Immunohistochemical results suggested that the caprine CAR bacillus was closely related to the rabbit CAR bacillus.

Immunohistochemical demonstration of Mycoplasma gallinarum and Mycoplasma gallinaceum in naturally infected hen oviducts.

Using indirect immunoperoxidase (IIP), peroxidase anti-peroxidase (PAP) and avidin biotin-peroxidase complex (ABC) immunohistochemical methods, Mycoplasma gallinarum and M gallinaceum antigens were demonstrated in ethanol-fixed paraffin-embedded oviduct sections from hens the eggs from which showed suboptimal hatchability. Specific immunoperoxidase staining was detected at the mucosa in the magnum portion of the oviduct. Optimal staining was achieved by applying the ABC method, though both IIP and PAP methods can also be used for diagnosis. Isolation and identification techniques gave similar results for the species of avian mycoplasmas involved.

Polyarthritis in kids associated with Mycoplasma putrefaciens.

Fifteen two- to three-week-old kids, fed artificially with goats' milk from a dairy, were found to have polyarthritis. The most affected joints were the carpals, either unilaterally or bilaterally, and in some cases the tarsal joints. Mycoplasma putrefaciens was isolated from the joints which showed an acute fibrinopurulent arthritis. No clinical mastitis was detected in the dairy.