Isolation and characterisation of bacteria associated with flying insects in hospitals, with particualr emphasis on Clostridum difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies.

Ornamental plants as climatic indicators of arthropod vectors

The importance and risk of vector-borne diseases (eg. leishmaniasis, West Nile Virus, Lyme borreliosis) is going to increase in the European temperate areas due to climate change. Our previous studies have shown that the potential distribution of Leishmania infantum and some Phlebotomus (sand fly) species – a parasite of leishmaniasis, and its vectors – may be expanded even to the southern coastline of the Baltic Sea by the end of the 21st century. The lowland areas of the Carpathian Basin and the main part of Hungary are projected to be suitable for the studied sand fly vectors in the near future. It is important to find some indicator plants to examine whether the sand flies are able to live in a certain climate at a certain time. We studied several Mediterranean and Sub-Mediterranean plant species, and we found that the aggregated distribution of three ligneous species (Juniperus oxycedrus L., Quercus ilex L. and Pinus brutia Ten.) shows high correlation with the union distribution of five sand flies (Phlebotomus ariasi Tonn., Ph. neglectus Tonn., Ph. perfiliewi Parrot, Ph. perniciosus Newst. and Ph. tobbi Adler, Theodor et Lourie). Since these Mediterranean species are highly tolerant of the edaphic characteristics of the planting site, they may prove to be good indicators. The present and upcoming climate of Hungary is seen to be suitable for the selected indicator plant species, and it draws attention to and verifies the potential of the expansion of sand flies, which has been proved by some recent observations of the vectors in Southern Hungary.

The combined impact of urban heat island, thermal bridge effect of buildings and future climate change on the potential overwintering of Phlebotomus species in a Central European metropolis

Leishmaniasis is one of the most important emerging vector-borne diseases in Western Eurasia. Although winter minimum temperatures limit the present geographical distribution of the vector Phlebotomus species, the heat island effect of the cities and the anthropogenic heat emission together may provide the appropriate environment for the overwintering of sand flies. We studied the climate tempering effect of thermal bridges and the heat island effect in Budapest, Hungary. Thermal imaging was used to measure the heat surplus of heat bridges. The winter heat island effect of the city was evaluated by numerical analysis of the measurements of the Aqua sensor of satellite Terra. We found that the surface temperature of thermal bridges can be at least 3-7 °C higher than the surrounding environment. The heat emission of thermal bridges and the urban heat island effect together can cause at least 10 °C higher minimum ambient temperature in winter nights than the minimum temperature of the peri-urban areas. This milder micro-climate of the built environment can enable the potential overwintering of some important European Phlebotomus species. The anthropogenic heat emission of big cities may explain the observed isolated northward populations of Phlebotomus ariasi in Paris and Phlebotomus neglectus in the agglomeration of Budapest.

Európai lepkeszúnyog fajok potenciális elterjedése a 21. században

Leishmaniasis is a typical vectorial disease transmitted by Psycodidae vectors (Lutzomyans, Phlebotomus species). The worldwide observed 1,5-2 million new cases and 60,000 death caused by Leishmania parasites per year make leishmaniasis is one of the most important vectorial disease in the tropicals and warm temperate areas of the World. In the human environment dogs and cats are the most important hosts of the different leishmania agents. The different leishmania species cause symptomatically cutan or visceral disease forms, but many other type of the disease has recognised. Phlebotomus species are sensitive to climatic patterns, they require hight relative air humidity, mild winters and long and warm vegetation period, but the environmental requirements of the species naturally is not the same. Due to climate change in the near future the climate of Western and Central Europe could allow the colonisation of these highly populated areas with also the vectors and the parasites. Our aim was to analyse the environmental patterns of the current distribution area of 8 important sand flies (P. ariasi, P. perniciosus, P. perfiliewi, P. papatasi, P. tobbi, P. neglectus, P. similis and P. sergenti) using the 1960-1990 period’s climate as reference. Using climate envelope modeling we determined these climatic characters and using the REMO climate projection we created the recent and the near-future (2011-2040 and 2041-2070) potential distribution area of the sand flies. The current known area of many Phlebotomus species restricted either to the western or to the eastern Mediterranean Basin. We found that their climatic requirements are could not explain their segregation, it is maybe the consequence of their evolutionary history (geographical barriers and paleoclimatic history). By the end of the 2060’s most parts of Western Europe can be colonized by sand flies, mostly by P. ariasi and P. pernicosus. P. ariasi showed the greatest potential northward expansion. Our model resulted 1 to 2 months prolongation of the potentially active period of P. neglectus P. papatasi and P. perniciosus for the 2070’s in Southern Hungary. As the climate becomes drier and warmer, sand flies will occupy more and more parts of Hungary. Our findings confirm the concerns that leishmanisais can become a real hazard for the major part of the European population to the end of the 21th century and the Carpathian Basin is a particularly vulnerable area.

Ornamental plants as climatic indicators of arthropod vectors

The importance and risk of vector-borne diseases (e.g., leishmaniasis, West Nile Virus, Lyme borreliosis) is going to increase in the European temperate areas due to climate change. Our previous studies have shown that the potential distribution of Leishmania infantum and some Phlebotomus (sand fly) species – a parasite of leishmaniasis, and its vectors – may be expanded even to the southern coastline of the Baltic Sea by the end of the 21st century. The lowland areas of the Carpathian Basin and the main part of Hungary are projected to be suitable for the studied sand fly vectors in the near future. It is important to find some indicator plants to examine whether the sand flies are able to live in a certain climate at a certain time. We studied several Mediterranean and Sub-Mediterranean plant species, and we found that the aggregated distribution of three ligneous species (Juniperus oxycedrus L., Quercus ilex L. and Pinus brutia Ten.) shows high correlation with the union distribution of five sand flies (Phlebotomus ariasi Tonn., Ph. neglectus Tonn., Ph. perfiliewi Parrot, Ph. perniciosus Newst. and Ph. tobbi Adler, Theodor et Lourie). Since these Mediterranean species are highly tolerant of the edaphic characteristics of the planting site, they may prove to be good indicators. The present and upcoming climate of Hungary is seen to be suitable for the selected indicator plant species, and it draws attention to and verifies the potential of the expansion of sand flies, which has been proved by some recent observations of the vectors in Southern Hungary.

Drosophila Flight Stabilization Depends on Apparent Distances in Optic Flow Field

To perform daily flight tasks, insects rely heavily on their visual perception of a dynamic environment. They must process visual signals quickly and accurately and update their behavior. Flies are vulnerable to environmental disturbances, such as gusts of wind blowing them off course, but they may use the altered visual field to compensate and regain their original course. In studies using Drosophila melanogaster, it has been shown that their corrective responses can be analyzed by measuring changes in their wing beats. By enclosing a tethered fly in a cuboidal visual arena displaying a computerized optic flow field, it is possible to calculate the change in wing beat amplitudes from an infrared shadow of its wings using photodiodes and a custom wing beat analyzer. In this experiment, manipulations ofthe optic flow field are used to create a field where points have varying relative forward speed, to study how the insect performs corrective maneuvers. The results show that Drosophila have a stronger corrective response to the quickly moving, apparently near points compared to the slower moving, apparently distant points. This implies the flies are distinguishing points based on their relative speeds, inferring distance, and adjusting their corrective actions with this information.

Understanding the Effect of Protein Tyrosine Phosphatase Receptor Type 0 on Mammalian Sensory Development: Behavioral Studies on PTPRO Knockout Mice

Nerve development, which includes axon outgrowth and guidance, is regulated by many protein families, including receptor protein tyrosine phosphatases (RPTP's).Protein tyrosine phosphatase receptor type 0 (PTPRO) is a type III RPTP that is important for axon growth and guidance, as observed in chicks and flies. In order to examine the effects ofPTPRO on mammalian development, standard behavioral tests were used to compare mice lacking the gene for PTPRO (ROKO mice) to wild-type (WT) mice. The ROKO mice showed a significant delay in reacting to a thermal noxious stimulus, hotplate analgesia, when compared to the WT mice suggesting deficient nociceptive function. In a rotarod test for proprioceptive function the ROKO mice exhibited a significant decrease in the amount of time spent on the rotating rod than did the WT mice. Additional proprioception tests were performed including the climb, step reflex, beam, and mesh walk tests. In the climb and step (place) test, the ROKO group had a significantly lower accuracy in performing the tests than did the WT mice. Thus, mice lacking the PTPRO gene showed behavioral deficiencies that reflect impairment in sensory function, specifically for nociception and proprioception.

EFFECTS OF A VISUAL HORIZON ON DROSOPHILA MELANOGASTER OBJECT-TRACKING RESPONSES

To navigate effectively in three-dimensional space, flying insects must approximate distances to nearby objects. Humans are able to use an array of cues to guide depth perception in the visual world. However, some of these cues are not available to insects that are constrained by their rigid eyes and relatively small body size. Flying fruit flies can use motion parallax to gauge the distance of nearby objects, but using this cue becomes a less effective strategy as objects become more remote. Humans are able to infer depth across far distances by comparing the angular distance of an object to the horizon. This study tested if flying fruit flies, like humans, use the relative position of the horizon as a depth cue. Fruit flies in tethered flight were stimulated with a virtual environment that displayed vertical bars of varying elevation relative to a horizon, and their tracking responses were recorded. This study showed that tracking responses of the flies were strongly increased by reducing the apparent elevation of the bar against the horizon, indicating that fruit flies may be able to assess the distance of far off objects in the natural world by comparing them against a visual horizon.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.

Proteomic Analysis of Wolbachia Symbiosis within the Drosophila

Wolbachia pipientis are bacterial endosymbionts of arthropods and in some filarial nematodes. Wolbachia are of particular interest because nematodeWolbachia have been shown to cause the diseases African river blindness and Lymphatic Filariasis. Doxycycline can be used to eliminate nematode Wolbachia, however, more efficient treatments are needed. Ideally, we would like to repurpose another FDA approved drug that helps to shorten treatment duration. Vitamins are one of the best classes of FDA approved compounds, generally recognized as safe. Interestingly, prior work by Serbus and colleagues found that dietary yeast, which is highly enriched in vitamins, dramatically reducesWolbachia titer in Drosophila melanogaster ovarian tissue. Imaging data indicated that the Wolbachia nucleoids were disrupted in response to yeast. This raised the possibility that yeast cells contain a bio-reactive, anti-Wolbachiacompound. Our close examination of yeast nutritional information identified which vitamins are most highly enriched in yeast. We then administered several of these to D. melanogaster, and saw that two of these led to reduced ovarianWolbachia titers, analogous to yeast-fed flies. This was especially interesting, as both vitamins are critical for functioning of the same biochemical pathway. We used retested effect of one of these vitamins in oogenesis by performing a dilution series, and achieved positive correlation from this dilution series. This opens up the avenue for clarifying the mechanism of how vitamins suppressWolbachia titer, and for testing enhancement of Doxycycline, to hopefully provide faster, more affordable treatment for millions of patients.

The 1994 BFA Graduating Class Departmrnt of Visual Arts

I find it hard to believe that already we are graduating our third group of students. It seem like our department was founded only a few months ago. Time Flies! Over the years I've come to respect the energy,Principles, commitment, talent and diversity of my colleagues in visual arts. I have never worked with such an outstanding group of people, and I firmly believe we deliver a University experience second to none. Having said that, I also believe our success would be diminished were it not for the fact that we have had the priviledge of working with students who were equally outstanding. Newfoundlanders can be justly proud of the Sir wilfred Grenfell College graduates from the many communities throughout the province who will represent Newfoundland and Labrador admirably on regional, national and international levels in years to come. This years catalogue records part of the many accomplishments of the class of 1994. Once again it is evident that discovering, nuturing and developing individual expression yields artwork of substance and consequence. It also confirms our belief that students should independent, resourceful and creative problem-solvers with a firm belief in their own view-points and the wherewithall to express those viewpoints through personal visual expression.

The housefly Musca domestica as a mechanical vector of Clostridium difficile

Background - Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. Aim - To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. Methods - M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. Findings - M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4 h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/− 66.9 for vegetative cell suspension and 288.2 +/− 83.2 for spore suspension). After 1 h, this had reduced (21.2 +/− 11.4 for vegetative cell suspension and 19.9 +/− 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/− 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/− 0.58. C. difficile could be recovered from fly excreta for up to 96 h. Conclusion - This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas.

The white gene and locomotor recovery from anoxia in Drosophila melanogaster

Locomotor recovery from anoxia is complicated and little is known about the molecular and cellular mechanisms regulating anoxic recovery in Drosophila. For this thesis I established a protocol for large-scale analysis of locomotor activity in adult flies with exposure to a transient anoxia. Using this protocol I observed that wild-type Canton-S flies recovered faster and more consistently from anoxia than the white-eyed mutant w1118, which carries a null allele of w1118 in an isogenic genetic background. Both Canton-S and w1118 are commonly used controls in the Drosophila community. Genetic analysis including serial backcrossing, RNAi knockdown, w+ duplication to Y chromosome as well as gene mutation revealed a strong association between the white gene and the timing of locomotor recovery. I also found that the locomotor recovery phenotype is independent of white-associated eye pigmentation, that heterozygous w+ allele was haplo-insufficient to induce fast and consistent locomotor recovery from anoxia in female flies, and that mini-white is insufficient to promote fast and consistent locomotor recovery. Moreover, locomotor recovery was delayed in flies with RNAi knockdown of white in subsets of serotonin neurons in the central nervous system. I further demonstrated that mutations of phosphodiesterase genes (PDE) displayed wild-type-like fast and consistent locomotor recovery, and that locomotor recovery was light-sensitive in the night in w1118. The delayed locomotor recovery and the light sensitivity were eliminated in PDE mutants that were dual-specific or cyclic guanosine monophosphate (cGMP)-specific. Up-regulation of cGMP using multiple approaches including PDE mutation, sildenafil feeding or specific expression of an atypical soluble guanylyl cyclase (Gyc88E) was sufficient to suppress w-RNAi induced delay of locomotor recovery. Taken together, these data strongly support the hypothesis that White transports cGMP and promotes fast and consistent locomotor recovery from anoxia.

The Role of G Protein In Alcohol Related Behaviours Using Drosophila melanogaster As A Model

Ethanol, classified as a drug, affects the central nervous system, and its consumption has been linked to the development of several behaviours including tolerance and dependence. Alcohol tolerance is defined as the need for higher doses of alcohol to induce the same changes observed in the initial exposure or where repetitive exposures of the same alcohol dose induce a lower response. Ethanol has been shown to interact with numerous targets and ultimately influence both short and long term adaptation at the cellular and molecular level in brain [1]. These adaptation processes are likely to involve signalling molecules: our work has focussed on G proteins gene expression. Using both wild type and several mutant fruit fly (Drosophila melanogaster) as a model for behaviour and molecular studies, we observed significant increases in sedation time (ST50) in response to alcohol (P<0.001) Fig.A. We also observed a consistent and significant decrease of Gq protein mRNA expression in Drosophila dUNC and DopR2 mutants chronically exposed to alcohol (*P<0.05). Fig B. Method: Six male flies were observed in drosophila polystyrene 25 x 95mm transparent vial in between cotton plugs. To the top plug, 500uL of 100% ethanol was added. Time till 50% of the flies were sedated was recorded on each day following the schedule. Fig. C (n=4-6). Using RT-PCR, we also quantified G protein mRNA expression levels one hour post initial 30 minutes of ethanol expression on day 1 and day 3 relative to expression in naïve flies.(n=2) [A] Increase in sedation time indicative of tolerance in different mutant lines and wild type flies. Six male flies were used in each experiment and (n= 4-6. ***P<0.001 unpaired t tests). [B] RT-PCR results showing significant reduction in Gq mRNA in flies chronically exposed to alcohol. (n=2. *P<0.05) [C] Alcohol exposure schedule. (1) Kaun K.R., R. Azanchi, Z. Maung, J. Hirsh, U. Heberlein. (2011). A Drosophila model for alcohol reward. Nature Neuroscience. 14 (5), 612–619.