Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates : Assay development for the determination of antibiotic susceptibility of Mycoplasma synoviae

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n=92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns

Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and timeconsuming isolation of M. synoviae strains

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine

: The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable

Sustaining peri-urban agriculture in rapidly urbanising cities in sub-Saharan Africa: A model and survey based assessment of adaptations to maintain peri-urban agriculture in Kumasi under threat of climate change and urban sprawl

Throughout sub-Saharan Africa, agricultural activities in urban and peri-urban areas are threatened by urban sprawl. These activities are important not just for the livelihoods of those who take part in it, but also for the food security of the city. Historically, in Kumasi 40% of the crops in the city are sourced from urban and peri-urban agriculture. However, Kumasi is also a city that is growing rapidly both in population and in area: since the 1980’s population has more than quadrupled and the area has increased more than tenfold. Because of this, there is a need to find ways to maintain urban and peri-urban agriculture under the threat of urban sprawl and the climate change in the future and also work well in the Kumasi context.We used the agro-hydrological model AquaCrop informed by 4.5 years of local weather data, soil data and crop data to model the effectiveness of adaptations in maintaining food security for three neighbourhoods in Kumasi. Local farmer management practices that were determined by a survey of 150 Kumasi farmers made a distinction in management practices between adaptations and crops. This model gave yields and irrigation water uses for five crop groups that are commonly cited as crucial for food security. Combined with population growth predictions and land use and land cover analysis this allowed us to make a statement about how well adaptations can meet current and future demand of crops and how much space is needed to meet demand. This model was supplemented with a survey of vendors and farmers to investigate barriers against- and preferences for adaptations.Backyard gardening and sack gardening turned out to be the best options to maintain peri-urban agriculture. These adaptations use the least space, for the highest yields. From the survey of farmers and vendors, no strong objections were found against the implementation of these adaptations. Over 80% of surveyed farmers and vendors felt that backyard gardening was a good adaptation to maintain peri-urban agriculture in Kumasi. Backyard gardening and sack gardening are optimally suited for growing vegetables and legumes, which are the easily perishable crops and thus benefit from a short supply line, which many vendors cite as solutions for spoilage.It is possible to ensure self-sufficiency for these crops with 5-9% of total land in the Feyiase neighbourhood, 11-20% in Ejisu and with 14-22% in the Kwadaso neighbourhood. It is therefore recommended to target at least a majority self sufficiency, by reserving a >50% fraction of this land for backyards and sack gardening spaces. At the same time, any available marginal lands should be allocated for the growth of cereals and tubers, to allow for the production of these subsistence crops, until the production of the remaining demand is fully met by rural import. With these adaptations and recommendations, it is possible for peri-urban agriculture in Kumasi to maintain its important role throughout the coming decades.In order to determine if there are any deviations from these conclusions, future research should focus on including empirical crop data tailored to AquaCrop or use a model that is better suited to represent the chaotic nature of (peri-) urban agriculture. While the model results do not differ significantly from the crop yields as found in literature, a model informed by local crop data can be an even better representation of the situation in Kumasi. Furthermore, a study into the long-term effects of adaptation on nutrition can reinforce our conclusions on food security.Finally, there is an opportunity to develop more high-tech agricultural methods like greenhouses and aquaponics. There is also an opportunity to incorporate wastewater reuse schemes, following examples from other countries, such a urine reuse, or low-tech treatment with sludge harvest. For this, it is recommended to set up educational programs and pilots.Civil Engineerin

Surface failures of plastically deformed rolling disks

Surface failures have been frequently observed in materials under conditions of cyclic rolling contact. The fatigue results from cumulative plastic deformation in a specific direction. Using the approximate theory established by Merwin and Johnson for a simplified model, an effort has been made to give certain predictions for the life to failure. Various quantities were taken into consideration as possible criteria for failure. The theoretical results were compared with experimental data. However, an insufficient number of experiments were performed to draw reliable conclusions from these data. The theoretical values did not give good agreement with an experimental law established by Palmgren. It was felt that for proper interpretation of the experimental data, several quantities' as for example the contact width, need to be measured which at this stage of the project was impossible