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The economic costs of contagious agalactia (CA) to the small ruminant dairy industry are not well known but include losses due to mortality, lowered milk production, spoiled products, abortions and animal welfare problems, as well as diagnosis and treatment. This paper reports financial estimates made in southern Europe, including a study on small- and large-scale farming systems in Italy, indicating that the financial losses are high and underestimated. Furthermore, the current control strategies, including chemotherapy and vaccination, in selected countries in Europe are described. In some countries, disease control is hampered by excessively strict veterinary legislation which discourages farmers and private veterinarians from notifying outbreaks because it leads to the prohibition of milk sales and can result in delays in lifting restrictions. In addition, new European Union legislation may downgrade the importance of CA, which will have implications for international research efforts. Finally, a series of recommendations are provided that cover the proper notification and handling of CA outbreaks, including movement control, current diagnostics, treatment, vaccination and disinfection.

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).

ResumenDespués de más de 40 años desde la descripción del Síndrome de Doble Compresión por Upton y McComas en el Lancet, sigue siendo un tema debatido que ha llevado a algunos cirujanos de mano a dudar de su existencia. La evidencia científica demuestra que la alteración del transporte axonal es el mecanismo más plausible en la fisiopatología de ese síndrome de compresión nerviosa. Se ha demostrado que una cuidadosa exploración clínica junto a un buen estudio neurofisiológico bien dirigido incrementa la eficiencia diagnóstica en términos de pacientes correctamente diagnosticados en el SDC. La liberación del Ligamento Transverso del Carpo está indicada en determinados pacientes que cumplen unos criterios de inclusión y exclusión específicos. Los resultados demuestran que la liberación del canal carpiano en esa población diana específica, es capaz de mejorar la gravedad de los síntomas de manera similar a la población afecta de Síndrome del Túnel Carpiano y de manera casi similar, a nivel de la Discapacidad de MMSS. Sólo instrumentos genéricos, como el SF-36, han sido capaces de detectar la diferencia a nivel de ciertas dimensiones de la Salud en relación a la Calidad de Vida entre los resultados del tratamiento en SDC y STC.

Due to their predatory habits, raptors may serve as indicators of the presence of antimicrobial‐resistant bacteria in the environment, but they also represent a public health risk for livestock and humans because they can act as reservoirs, sources and spreaders of these bacteria. Our objective was to determine the presence of antimicrobial‐resistant bacteria in cloacal samples of Canarian Egyptian vultures (Neophron percnopterus majorensis), an endemic bird of prey. One hundred and forty‐two cloacal swabs were obtained; Escherichia coli was isolated from 80.28% and Salmonella from 6.3% of these samples. Low levels of susceptibility to ampicillin, tetracycline and trimethoprim/sulfamethoxazole were found. About 20% of the isolates were resistant or presented intermediate susceptibility to fluoroquinolones. Surprisingly, we found isolates resistant to imipenem (6.96%). Isolates from chicks were more susceptible to antimicrobial drugs than adult and immature birds. About 50% of E. coli isolates were resistant to ampicillin, tetracycline and trimethoprim/sulfamethoxazole, and about 20% to piperacillin, enrofloxacin and marbofloxacin. High percentages of isolates of Salmonella were found to be resistant to cephalexin (88%) and aminoglycosides (greater than 77%). Our results support the idea that raptors could act as reservoirs of Salmonella and antimicrobial‐resistant bacteria, posing a risk not only to wildlife but also to livestock and the human population, thus reinforcing the need to minimize the exposure of wildlife to antimicrobial agent through human and livestock waste

The aim of this study was to compare PCR tests for the detection of Mycoplasma agalactiae in sheep and goats. Samples obtained from sheep and goats in Turkey were tested to determine the presence of M. agalactiae. Mycoplasma culture and two direct PCR methods based on the uvrC gene and polC gene were compared with PCR of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (PCR/DGGE) on DNA extracted from clinical samples (following culture enrichment). A total of 234 samples were examined: 9.4% and 13.2% were positive for M. agalactiae based on the uvrC gene and polC gene PCRs, respectively, while the culture method revealed that 12.8% of the samples were mycoplasma-positive. The PCR/DGGE method identified M. agalactiae in 15.0% of the samples; it also detected and identified other Mycoplasma species in the samples. M. agalactiae was only detected in goats in this study. This is the first time 16S rRNA PCR/DGGE was performed on various samples from sheep and goats in Turkey. This study confirmed that M. agalactiae and other Mycoplasma species are present in goats, indicating a requirement for effective control strategies for contagious agalactia and Mycoplasma species in Turkey.

The aim of the study was to evaluate the presence of Mycoplasma bovis infection and co-infections with other Mycoplasma spp. infections in cattle. The tested population was one in the eastern region of Poland containing 66 dairy cows and 23 calves showing different clinical signs and suffering from pneumonia, mastitis, and arthritis. The incidence of M. bovis in co-infections with other Mycoplasma spp. was examined using serological traditional mycoplasma culture methods, and the molecular methods - PCR and polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE). The PCR/DGGE method for detecting Mycoplasma spp. in cattle was used for the first time in Poland. The seroprevalence of M. bovis in the affected cattle herds in the eastern region of Poland was 47.8% in calves and 19.7% in dairy cows. The direct detection and identification of M. bovis from nasopharyngeal swabs by PCR revealed that 56.5% of calves were positive, but all of the dairy cows were negative. The PCR/DGGE identified eight (34.8%) instances of M. arginini and eight (26.1%) instances of M. bovirhinis co-infecting with M. bovis in ten calves. The seroprevalence of M. bovis in the tested population was 33.7%. Any future attempts to control mycoplasma infections require an insight into the current epidemiological situation of M. bovis infection and its relationship to other mycoplasmas in causing clinical disease in cattle. Using these diagnostic methods we have demonstrated that mycoplasmal infections are often caused by multiple species of Mycoplasma and not just the primary M. bovis pathogen.

Streptococcus suis is a recognized pathogen that may cause important diseases in pigs and humans. This microorganism has been repeatedly isolated from wild boar (Sus scrofa). However, its health implications for this wild species are still unknown. This article reports a detailed description of a fatal case of septicemia by S. suis affecting a young wild boar. The affected animal, about 15 days old, was found near death and exhibiting neurologic signs at a wild boar estate in southwestern Spain. Postmortem examination showed generalized congestion, brain hemorrhages and lobular pneumonia. Histopathological evaluation demonstrated the presence of meningitis and encephalitis with marked congestion and suppurative bronchopneumonia. Streptococcus suis serotype 2 isolates exhibiting important virulence factors (extracellular factor, muramidase-released protein, and suylisin) were isolated from the affected animal. This study confirms the presence of potentially virulent and zoonotic strains of S. suis in wild boar from Spain.

Changes in peripheral blood leukocyte subpopulations were investigated in calves challenged intratracheally with three different Mycoplasma bovis isolates in Groups E1, E2, and E3. The controls received a placebo. Blood samples were collected before challenge and then at days 1 to 7, 14, 21 and 28. White blood cells (WBC), polymorphonuclear leukocytes (PMNLs), lymphocytes (LYMs), monocytes, eosinophils and basophils (mid-size cells, MID), as well as CD2+, CD4+, CD8+, WC4+ lymphocyte subsets with CD4:CD8 ratio were also analysed. A transient increase of WBC and PMNLs in all challenged calves was observed on day 1. Increased LYM counts were observed in E1 throughout the study, whereas in E2 the LYM counts were higher only between days 14 and 28, and consistently lower in E3. The MID count had broadly comparable values for all groups. Stimulation of the CD2+ response was observed in E2 and E3 in contrast with E1 which had a lower CD2+ throughout. The CD4+ response was dominant in E1 and E2, whereas in E3 a parallel CD4+ and CD8+ stimulation was observed. The B-cell response (WC4+) and an increased CD4:CD8 ratio was most apparent in E1. The main host responses to M. bovis infections are a stimulation of CD4+ cells and an enhancement of the WC4+ response.

The presence of infection with Mycoplasma species in association with lung consolidation, environmental temperature and relative humidity was investigated in 410 clinically healthy fattening lambs from five different feedlots in Extremadura (southwestern Spain). Isolates of Mycoplasma species were obtained (n= 117), including Mycoplasma ovipneumoniae (n = 18) and Mycoplasma arginini (n = 99). Two seasonal periods were identified. The first period, which included February, March, September, October, and November, had an average temperature of 17.5 ± 4.7 °C and a relative humidity of 61.3 ± 15.8%. The second seasonal period, which included the months from April to August, had an average temperature of 22.9 ± 5.5 °C and a relative humidity of 48.4 ± 10.7%. Most Mycoplasma species were isolated from the second seasonal period, indicating that higher temperatures and lower relative humidity favour the presence of Mycoplasma species. M. arginini was also associated with lung consolidation.

Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected ( dnaA , metS , recA , tufA , atpA , rpoD , and tkt ). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.

Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis. Two avian pathogens, M. gallisepticum and M. synoviae have been isolated from the brains of poultry showing meningeal vasculitis and encephalitis. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain.

Aim To isolate and characterize strains of Mycoplasma agalactiae from bulk tank and silo ewes' milk. Methods and Results Thirteen mycoplasma isolates were obtained from samples of sheep milk taken from bulk tank and large silos and identified as Myc. agalactiae by PCR-DGGE. The isolates were typed by pulsed field gel electrophoresis (PFGE), SDS-PAGE and immunoblot. The in vitro activity of 13 antimicrobials of veterinary interest was tested against these isolates. Results showed that the most effective compounds against Myc. agalactiae in vitro were clindamycin, an antibiotic not previously described as a suitable contagious agalactia (CA) treatment, with Minimum Inhibitory Concentration (MIC) values of

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 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...

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.

International audience; AbstractPorcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215–06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.

The UK has approximately 10 million cattle (Veterinary Medicines Directorate 2011), and each year approximately 1.9 million of these cattle are affected by respiratory disease. A disease that is often complex, caused by viruses and/or bacteria, with as many as one-third of these infected with the Mycoplasma bovis pathogen (Nicholas and Ayling 2003). The welfare and economic effects of clinical cases of M. bovis on farming are therefore substantial and costs may be even more when morbidity associated with respiratory infection is considered. Apart from respiratory disease, M. bovis is also associated with other clinical signs, including mastitis, arthritis, meningitis, infertility, abortion and keratoconjunctivitis (Nicholas and Ayling 2003). Elimination of M. bovis is difficult and treatment with antimicrobials often has limited effect unless animals are treated early in the course of disease. In recent years, as much as 11 tonnes per annum of active antimicrobials have been sold for use in the UK cattle antimicrobial products, which includes all intramammary products (Veterinary Medicines Directorate 2011). With no commercial vaccines available in Europe, although use of autogenous vaccines has shown some success (Nicholas and others 2006), it is important to effectively target antimicrobial treatment to ensure prudent use of antimicrobials and to reduce the development of antimicrobial resistance. Between 2004 and 2009, in vitro minimum inhibition concentration (MIC) data for 45 M. bovis isolates have been determined for up to 13 antimicrobials. Forty-five epidemiologically unrelated M. bovis isolates from cattle were tested, as well as the National Collection of Type Cultures (NCTC) type strain (NCTC 10131); details are given in Table 1. Samples were previously submitted to the Animal Health and Veterinary Laboratories Agency for mycoplasma detection and identification by culture and molecular methods that include PCR and denaturing gradient gel electrophoresis (McAuliffe and others 2005). Isolates were subsequently stored …

Objectives M ycoplasma mycoides subspecies capri is one of the causative agents of contagious agalactia in goats. The disease is characterised by mastitis, pneumonia, arthritis, keratitis and in acute cases septicaemia. No vaccine is currently available that has been demonstrated to prevent disease. Methods This study used two-dimensional electrophoresis to separate proteins from whole-cell preparations and tandem mass spectrometry to identify them. Key findings In total, 145 spots were successfully identified corresponding to 74 protein identities. Twenty of these proteins were found to be immunogenic by western blot analysis using a pooled serum sample from experimentally infected goats. Conclusions Six proteins were found to have a less than 95% amino acid similarity to a closely related Mycoplasma species showing that they warrant further evaluation in development of diagnostic tests. These proteins were a dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex, phosphoglycerate kinase, pyrimidine-nucleoside phosphorylase, 30S ribosomal protein S6, ribulose-phosphate 3-epimerase and D-lactate dehydrogenase.

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.

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.

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.

Flow cytometry has become a valuable tool in different fields of microbiology, such as clinical microbiology, aquatic and environmental microbiology, food microbiology, and biotechnology. It combines direct and rapid assays to determine numbers, biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population. This review focuses on the applications of flow cytometry to the field of mycoplasmology. It tries to give a scope of the important breakthroughs which occurred in this field in the last decades, and in the advantages of introducing flow cytometry in research and routine diagnostic procedures of mycoplasmas.

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.

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.

Mycoplasmas are the smallest and simplest organisms known. They form a large group of bacteria that can infect humans, animals, and plants. Even though several techniques have been proposed to enumerate mycoplasmas in broth medium, the determination of mycoplasma growth still remains a difficult task. The potential of using flow cytometry (FC) for rapidly estimating several species of mycoplasmas, M. agalactiae (Ma), M. putrefaciens (Mp), M. capricolum subsp. capricolum (Mcc), M. bovis (Mb), M. capricolum subsp. capripneumoniae (Mccp) and M. hyopneumoniae (Mh) in broth medium was examined. The FC analysis was performed by staining the mycoplasma cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count (Colony Forming Units--CFU) or Colour Changing Units (CCU) methods, depending on the mycoplasma species. There was a good correlation between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count (CFU) or CCU methods. A correlation of 0.841, 0.981, 0.960, 0.913, 0.954, and 0.844 was obtained for Ma, Mp, Mcc, Mb, Mccp and Mh, respectively. FC method allowed results in 20-30 min, while 24-72 h was necessary for plate count method and 15 days for CCU method. FC was found to be a very useful, practical and fast technique to count mycoplasmas. These findings suggest that FC can be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasmas in broth medium.