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Summary Mycoplasma pneumoniae (Mp) is a leading cause of human community-acquired pneumonia. To investigate the pathogenesis of the infection, 36 gerbils were intranasally inoculated with Mp culture (30 animals) or sterile mycoplasma broth (6 animals) and euthanized from 1 to 5 weeks post infection. A morphological and immunohistochemical study was carried out in all animals to determine the cellular populations present in lung parenchyma. Polyclonal and monoclonal antibodies were used to detect antigens of Mp and CD3, CD4, CD8 and CD79 lymphocytes, as well as cells containing S100 and major histocompatibility complex class II (MHC-II) antigens. There was progressive infiltration of mononuclear cells in the lamina propria of bronchi and bronchioles, and hyperplasia of the bronchus-associated lymphoid tissue (BALT) in the infected animals. BALT contained dendritic cells immunopositive to S100 and MHC-II and numerous CD3, CD4 and CD79 lymphocytes. The immunohistochemical results showed that T lymphocytes, particularly CD4 and CD79 cells, may play a role in the immune response of gerbils against Mp. This experimental model is valuable for investigation of the pathogenesis of Mp infection and may assist in the development of therapeutic strategies.

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PET (DSM 105487T, CIP 111404T) and 5HT (DSM 105,488T, CIP 111405T), respectively., RRM acknowledges the Spanish Ministry of Economy project CLG2015_66686-C3-1-P also supported with European Regional Development Fund (FEDER) funds. ASR acknowledges the Gobierno de Canarias (Spain) project P2007/046. RRM acknowledges the financial support of the sabbatical stay at Georgia Institute of Technology supported by the grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1,759,831).

BackgroundMDR bacterial infections are currently a serious problem for clinicians worldwide. Klebsiella pneumoniae and Enterobacter spp., among Enterobacteriaceae, and Pseudomonas aeruginosa, are part of the group of ESCAPE pathogens or bacteria that ‘escape’ from common antibacterial treatments. The lack of effectiveness of the first common line of antibiotics has led to the search for new therapies based on older antibiotics, such as colistin.ObjectivesWe searched for new enhancers of the action of colistin against MDR Gram-negative bacteria that can be easily applicable to clinical treatments.MethodsColistin MICs were determined alone and with the protonophores CCCP, sodium benzoate, sodium salicylate and aspirin using the broth microdilution method and FIC indexes were calculated to assess synergy between colistin and each chemical. Time–kill assays of colistin with and without protonophores were performed to determine the bactericidal action of combinations of colistin with protonophores. Likewise, the effect of sucrose, l-arginine and l-glutamic acid on the MICs of colistin alone and combined with each protonophore was assessed.ResultsIt was found that sodium benzoate, sodium salicylate and aspirin, at concentrations allowed for human and animal use, partially or totally reversed resistance to colistin in P. aeruginosa and highly resistant enterobacterial strains. The mechanism of action could be related to their negative charge at a physiological pH along with their lipid-soluble character.ConclusionsSodium benzoate, sodium salicylate and aspirin are good enhancers to use in antibiotic therapies that include colistin.

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.

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.

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.

Caprine contagious agalactia is a syndrome most frequently caused by Mycoplasma agalactiae. The pathogenic mechanisms that allow M. agalactiae to persist in the mammary gland tissues following infection, despite a prominent inflammatory response, are yet to be fully established. The aim of the present study was to investigate cyclooxygenase (COX)-2 in the mammary gland of goats during M. agalactiae infection. COX-2 expression was evaluated by immunohistochemistry in the inflammatory lesions of 10 goats affected with M. agalactiae-induced mastitis (five naturally infected and five experimentally infected). Epithelial cells, macrophages, endothelial cells, fibroblasts and, to a lesser extent, neutrophils demonstrated positive immunostaining for COX-2, associated with areas of mastitis and with the presence of M. agalactiae antigen. These research findings suggest that COX-2 is involved in the inflammatory response that occurs in caprine contagious agalactia.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field infected plant samples showed a single peak at 82.5 degrees C (+/- 0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 degrees C. Absolute quantification of phytoplasma titer was calculated using standard, curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 10(6) to 10(3) according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10(-6) for conventional PCR and 10(-7) for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material. (C) 2017 Elsevier Ltd. All rights reserved

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.

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 24097T = ATCC BAA-2157T). The antiserum of strain G.A.T has been deposited in the Mollicutes collection at Purdue University (Indiana, USA), This work was supported by the Ministerio de Educación y Ciencia Proyecto (AGL2006-13743/GAN), the Animal Health and Veterinary Laboratories Agency, Weybridge, UK, and the Institut Mediterrani d’Estudis Avançats CSIC-UIB, Spain. R. R. M acknowledges the support of the Spanish Ministry of Science and Innovation (projects CLG2009_12651-C02-01, CLG2009_12651-C02-02 and the Consolider Ingenio 2010 CE-CSD2007-0005), all three co-financed with FEDER support from the European Union

The role of inapparent carriers of Mycoplasma agalactiae and the strategies used to colonise the external ear canal in goats remain unclear. This study examined the ability of M. agalactiae to colonise the ears of goats infected experimentally by the intramammary route. The right mammary glands of 15 lactating goats were inoculated with 10(10) colony forming units (cfu) of M. agalactiae. The goats were randomly assigned to three groups of five animals each and sampled at slaughter at 5, 15 or 45 days post-infection (dpi). A further four goats served as uninfected controls. Right and left ear swabs were collected for detection of M. agalactiae by culture before and after sacrifice. M. agalactiae was detected in 19/20 (95%) ear swabs from goats sampled at 15 and 45dpi, whereas all ear swabs collected before inoculation, ear swabs collected from the group sampled at 5dpi and ear swabs from control goats at the time of sacrifice were negative for M. agalactiae. Blood samples collected at 6, 12, 24, 48 and 72h post-infection for detection of M. agalactiae by culture were also negative. There were differences in the antigenic profiles of isolates recovered from the ears compared to the 7MAG strain used to inoculate the animals and most isolates from the mammary gland, milk and supramammary lymph nodes.

M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66% of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba.

Mycoplasma bovis is a serious, worldwide-spread but often overlooked pathogen causing respiratory disease, mastitis, and arthritis in cattle. In this study we characterize the protein and antigenic profiles of M. bovis field strains isolated in Bosnia and Herzegovina by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, and analyze possible variations among these strains. Greater differences occurred when comparing field strains with the reference strain PG45. One field strain isolated from lung samples of a heifer was markedly different from strains isolated from nasal swabs taken from cattle raised in another geographic region. A possible correlation may exist between protein and antigen profiles of M. bovis field strains, geographic regions and anatomical sites of isolation. Mycoplasma bovis, cattle, antigen, immunoblotting, protein, SDS-PAGE Mycoplasma bovis is the most pathogenic bovine mycoplasma in the parts of the world considered to be free of contagious bovine pleuropneumonia. This pathogen primarily causes calf pneumonia, mastitis and arthritis. Other symptoms are seen less frequently. Diseases caused by M. bovis are found worldwide, causing significant economic losses. Yet, there are no effective control measures or standard protocols for their routine application available (Nicholas and Ayling 2003). Although M. bovis, like other mycoplasmas, possesses a small genome size, it is still highly invasive and able to colonize organ systems. It is now clear that one of the possible explanations for its ability to avoid bodily defence mechanisms and adapt to different environments in the host is due to phenotypic variations in mycoplasmas (Wise 1993; Razin et al. 1998; Minion 2002). Rosengarten et al. (1994) demonstrated highly variable membrane surface proteins (Vsps) of M. bovis which represent a family of antigenically and structurally related lipoproteins. These Vsps represent the predominant antigens recognized by the host immune response during infection, and they can undergo high frequency changes in size and expression. This system is responsible for the survival of these pathogens in the presence of cytolytic antibodies and for their adaptation to specialized environments within their respective hosts (Behrens et al. 1994; Razin et al.1998). A full understanding of phenotypic variation among M. bovis strains should be of great value when developing diagnostic methods and vaccines for the control of infections caused by this organism. Recently, M. bovis has been isolated, for the first time, in Bosnia and Herzegovina (Rifatbegovic et al. 2007). The aim of this study was to characterize the protein and antigenic profile of these M. bovis field strains by SDS-PAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis) and immunoblot and to analyze possible variations among these strains.

The composition of the medium used to cultivate Mycoplasma species is very important. Serum is one of the most important additives as it contains lipids (cholesterol) and serum proteins, which are essential for the growth of the organisms. This work reports the development of a semi-defined medium, called MWS (Medium Without Serum) produced without animal serum and bovine serum albumin. MWS seems to be suitable for cultivating several species of caprine mycoplasma, especially M. mycoides subsp. mycoides (LC) and M. mycoides subsp. capri.

Assuncao, P., Machado, M.P., Fe, CD., Ramirez, A.S., Rosales, R.S., Antunes, N.T., Poveda, C. and Poveda, J.B. 2007. Prevalence of pathogens in great white pelicans (Pelecanus onocrotalus) from the Western Cape, South Africa. J. Appl. Anim. Res., 32: 29–32. Great White pelicans (Pelecanus onocrotalus) breed in Africa, Europe and Asia from Greece to Vietnam. In Africa, the Western Cape is probably the only place in the world where pelican numbers have shown a sustained increase over the past few decades. Nothing is known regarding the prevalence of pathogens present in these populations. Therefore, 50 Great White Pelicans from Western Cape were tested for the presence of various bacteria and viruses by polymerase chain reaction (PCR). It was observed that 49 pelicans were positive for Mycoplasma spp., 22 for Salmonella spp. and 3 for NDV, making these a potential risk to domesticated avian species, as well as human beings. This information is to be considered while planning any expansion/control p...

Contagious agalactia (CA) is an infectious syndrome caused by several species of mycoplasma and classically characterized by the triad of mammary, joint and eye symptoms, although further symptoms may also appear. CA is the most serious disease of whatever they affect in small dairy ruminants and so there is an urgent need for administrative bodies and farmers to join forces and design serious strategies with the final aim of completely eradicating the disease. CA is endemic in the Mediterranean and certain Asian and African regions, while in America it is described as sporadic. The emergence of CA in a non-infected herd is almost always linked to the introduction of carriers of CA or to contact with infected herds. It normally implies the rapid spread of clinical problems in the herd coinciding with the period of parturitions and the start of lactation. In endemic areas, this disease provokes subclinical or mild clinical symptoms, and the lack of an established, systemized diagnostic procedure implies, that many infected herds pass unnoticed and may act as a permanent source of infection. Enzyme-linked immunosorbent assay (ELISA) techniques are recommended for the detection of antibodies, but the systematic vaccination of herds in endemic areas prevents the use of serology to detect infected herds. Then, culture and polymerase chain reaction (PCR) techniques must be used. In clinical outbreaks, control measures should be directed towards limiting the economic losses of the herd and avoiding the spread of the disease by isolating the infected herd, antibiotic treatment, selective culling, disinfection measures and the classic prevention measures for mastitis (correct functioning and maintenance of milking equipment, the milking routine, teat dipping, etc.). In chronically infected herds, control measures should be directed to restrict disease spread by way of prevention measures for mastitis, detection and culling of animals with lesions, the administration of antimycoplasma drugs to all animals at the end of lactation, adequate management of replacement animals and, perhaps, vaccination.

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 ,o rMycoplasma 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 10 3 –10 4 cells ml 21 . 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. ' 2007 International Society for Analytical Cytology

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.

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.

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, r2 = 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 104 cells ml−1 and 103 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).

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.

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).

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.

Assuncao, P., Fe, C. de la, Ramirez, A.S., Sarradell, J.E. and Poveda, J.B. 2004. Characterization of a Mycoplasma agalactiae strain, candidate to an attenuated vaccine. J. Appl. Anim. Res., 26: 1–5. The aim of this work was to study the stability of a M. agalactiae attenuated strain after lyophilization and its characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Immunoblot against homologous antisera showed that the attenuated M. agalactiae strain had 2 characteristic antigenic bands of 32 and 21 kDa. The assays related to strain lyophilization showed that the CFUs were decreased lesser than 2log10 after 1 year storage at 4C with the exception of one vial, which lost all its viability. The subcutaneous inoculation dose (105 CFU), did not produce any symptom or sign of disease during the 45 days of the experiment. However, a local reaction was observed on the day after the inoculation. At necropsy, no macroscopic lesions were found which re...

Mycoplasma putrefaciens is listed as one of the etiologic agents of the contagious agalactia syndrome by the world organisation for animal health. This species has been characterized only recently, 1974, and the number of outbreaks caused by this microorganism so far is very scarce. It induces mastitis in infected goats although other symptoms such as arthritis in adults and septicaemia in kids are also frequently described. Up to now, the identification of M. putrefaciens relied on classical isolation and identification techniques which present a number of limitations. Specific primers for PCR have been designed based on sequence comparisons of the Arc B gene among the 'Mycoplasma mycoides cluster' and related species such as Mycoplasma cottewii and Mycoplasma yeatsii. Sequence alignments confirmed the taxonomic position of M. putrefaciens, which is related to the W. mycoides cluster' but also very close to M. yeatsii. The polymorphism observed amongst the different Arc B sequences allowed the determination of a primer pair yielding a specific amplification of a 316 bp-long DNA fragment by PCR. This PCR was validated in two different laboratories with a variety of mycoplasma strains isolated from goats. This new PCR technique will be very useful for a quicker determination of M. putrefaciens strains as well as a better understanding of the prevalence of M. putrefaciens infections. (Resume d'auteur)

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 MIC 50 and MIC 90 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 MIC 50 and MIC 90 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 MIC 50 and MIC 90 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.

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.

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.

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.

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.

The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp. mycoides large-colony type (LC) strain Y-goat and the M. mycoides subsp. capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been named lppA [MmymyLC] and lppA [Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp. mycoides small-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplified lppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the other members of the M. mycoides cluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.

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 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 mechanisms responsible for the increase in ceftazidime MIC in two Escherichia coli in vitro selected mutants, Caz/20-1 and Caz/20-2, were studied. OmpF loss and overexpression of acrB, acrD and acrF that were associated with acrR and marR mutations and sdiA overexpression, together with mutations A233T and I332V in FtSI (PBP3) resulted in ceftazidime resistance in Caz/20-2, multiplying by 128-fold the ceftazidime MIC in the parental clinical isolate PS/20. Absence of detectable β-lactamase hydrolytic activity in the crude extract of Caz/20-2 was observed, and coincided with Q191K and P209S mutations in AmpC and a nucleotide substitution at −28 in the ampC promoter, whereas β-lactamase hydrolytic activity in crude extracts of PS/20 and Caz/20-1 strains was detected. Nevertheless, a fourfold increase in ceftazidime MIC in Caz/20-1 compared with that in PS/20 was due to the increased transcript level of acrB derived from acrR mutation. The two Caz mutants and PS/20 showed the same mutations in AmpG and ParE.

In a herd of 200 Spanish sheep, 20% of the animals developed unilateral or bilateral keratoconjunctivitis, affecting young and adult animals. M. agalactiae was isolated from the conjunctival sac of 20 adult sheep and three lambs. After a topical and parenteral treatment using tetracycline and tylosine, all animals gradually recovered. One sheep was sacrificed to carry out a pathological study of the cornea, where mycoplasma were seen ultrastructurally. After 3 months of clinical presentation, M. agalactiae was again isolated from healthy sheep confirming the persistence of this microorganism.

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.

Organisms with the typical characteristics of mycoplasmas were isolated from the tracheae of buzzards, saker falcons, and griffon vultures. All isolates obtained from the same bird species appeared to be serologically identical and distinct from the isolates obtained from the other two bird species and from the previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that the isolates belong to three new species, for which the names Mycoplasma buteonis (seven strains obtained from buzzards), Mycoplasma falconis (six strains obtained from saker falcons), and Mycoplasma gypis (eight strains obtained from griffon vultures) are proposed. M. buteonis ferments glucose, does not hydrolyze arginine or urea, reduces tetrazolium chloride and potassium tellurite anaerobically, does not produce films and spots, and does not possess phosphatase activity. M. falconis does not ferment glucose, hydrolyzes arginine but not urea, reduces tetrazolium chloride and potassium tellurite anaerobically, does not produce films and spots, and does not possess phosphatase activity. M. gypis does not ferment glucose, hydrolyzes arginine but not urea, reduces tetrazolium chloride and potassium tellurite aerobically and anaerobically, produces films and spots, and possesses phosphatase activity. The three new species lyse avian, bovine, equine, human, ovine, porcine, rabbit, and guinea pig erythrocytes, but do not adsorb these erythrocytes. The type strain of M. buteonis is Bb/T2g (= ATCC 51371), the type strain of M. falconis is H/T1 (= ATCC 51372), and the type strain of M. gypis is B1/T1 (= ATCC 51370).

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, b-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 b-lactamase activity. Likewise, active efflux associated with increased levels of acrB and tolC and decreased OmpFexpression 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.

M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66% of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba.

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 goat's mammary gland.

A total of 120 mycoplasma strains were recovered from 97 of 265 diseased seals investigated during the seal epidemic in the North Sea and in the Baltic Sea in 1988. Mycoplasmas were isolated from the respiratory tracts (including lungs), hearts, brains, and eyes of the seals. Thirty strains were filter cloned and investigated for their morphological, biochemical, and serological characteristics compared with the characteristics of previously described species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that these strains belong to two new species, for which the names Mycoplasma phocarhinis and Mycoplasma phocacerebrale are proposed. M. phocarhinis (17 strains) did not ferment glucose or hydrolyze arginine but did reduce tetrazolium chloride and potassium tellurite and produced films and spots. M. phocacerebrale (13 strains) metabolized arginine but not glucose and produced phosphatase but did not reduce tetrazolium chloride and potassium tellurite. Both species lysed sheep erythrocytes but did not absorb sheep or guinea pig erythrocytes. The type strain of M. phocarhinis is strain 852 (= ATCC 49639), and the type strain of M. phocacerebrale is strain 1049 (= ATCC 49640).

We describe three different outbreaks of mastitis caused by M. mycoides subspecies mycoides LC type (Mmm LC) in three goat flocks from the Extremadura Region of south-west Spain. Thirty-two fast-growing isolates were obtained on Hayflick's and Friis's media with inhibitors from different specimens. All were identified as Mmm LC in spite of their cultural, biochemical and serological features.