Bacterial Vector: Delivery of Plasmid Mediated DNA Vaccine

Bacterial Vector: Delivery of Plasmid Mediated DNA Vaccine Abstract / Introduction: The United Nations recently estimated that the worlds population has exceeded seven billion people. It is projected that approximately 16% of this population rely on fish as a principal source of the protein obtained from animals [1]. However, many wild stocks of fish have begun to collapse due to destructive overfishing and damage caused to aquatic ecosystems by climate change. Aquaculture is the farming of aquatic species under environmentally managed conditions and is increasingly used throughout the world as a major food production method, providing approximately half of the fish consumed globally [3]. Sustainable aquaculture will play an important role in meeting the food requirements of a growing population while reducing the impact commercial fishing plays on overexploited and endangered species. Of critical importance when raising aquatic organisms under densely populated conditions is disease prevention. In an aquaculture setting with large populations of fish in close quart ers, bacterial and viral pathogens can spread rapidly partially due to the efficacy of transmission in water [4]. Therefore, an important consideration in moving towards sustainable aquaculture is effective and efficient prophylactic methods for preventing infection. Reduction of labour and material costs is of major concern in aquaculture disease management, therefore manual vaccination methods, such as intramuscular injection, are not considered viable in this regard. Salmon are a fish species of major commercial aquaculture relevance and as such, disease prevention in these farming scenarios is of substantial economic and environmental concern [5, 6]. One disease of relevance to farmed salmon species is the infectious hematopoietic necrosis virus (IHNV), a.k.a. Chinook salmon disease. It is a rhabdovirus whose genome encodes a glycoprotein, which presents as a viral antigen [7, 8], and is a pathogen that causes deadly disease in many salmonid species of fish. Since its discovery in the 1950s, IHNV has spread throughout North America, as well as to Asian and European countries. It causes necrosis and hemorrhage within infected fish, commonly in the kidney and spleen, and induces high mortality in young fry [9]. It can be particularly devastating in the densely populated aquaculture setting. Given the environmental sustainability and economic importance of commercially farmed salmon globally, it is therefore of importance to develop practical cost- effective methods for vaccinating large populations of fish against diseases such as IHNV. Gene based vaccines have been shown to be able to deliver plasmid-encoded DNA (pDNA) to fish cell cultures in vitro [10]. The protein produced inside the animal cell is treated as a foreign antigen, and can cause a protective immune response against a pathogen such as a bacteria or virus. Objectives / Methodology: The aims of this proposed research program are several fold. The objectives are to develop and test an appropriate bacterial vector for the delivery of a plasmid mediated DNA vaccine in an aquaculture environment. This vector will be assessed using molecular methods for efficacy within a suitable in vitro model system to examine the ability to deliver an immunologically relevant product of interest. This system will then be evaluated for effectiveness against pathogenic challenge within an appropriate in vivo model system. The hypothesis driving this proposal is that an engineered bacterial vector can effectively deliver a plasmid mediated gene vaccine within salmonid, and protect against a lethal challenge of a species and commercially relevant pathogen. Previous studies suggest that this is a worthwhile and meaningful pursuit in the global aquaculture context [11, 12]. Effective DNA vaccines against IHNV have previously been developed, however these types of vaccines have been hist orically delivered by intramuscular injection [11]. Several alternative methods of DNA vaccine delivery have been explored for including liposomes and ultrasound [13]. Though these methods present issues such as training, equipment costs, and sub-optimal vaccine delivery. An ideal scenario in an aquaculture context would involve a relatively inexpensive delivery vector (e.g. bacteria) carrying a vaccine, which can be easily dosed directly into the environment and induce a protective immunity within the population. The concept of using attenuated bacteria as DNA delivery vectors has been explored for some time [14, 15]. In prior studies, Escherichia coli have been shown to be capable of successfully acting as DNA delivery vectors to mammalian cells in vitro [12, 16]. For aquaculture settings, employing pathogens of human concern, even if attenuated, have regulatory and health concerns. For this reason delivery vectors such as E. coli are not ideal. In this study, an attenuated version of the salmonid pathogen Yersinia ruckeri, the cause of enteric redmouth disease (ERD) will be utilized. The rationale for using this attenuated vector is that it is a naturally occurring fish pathogen, in addition, it is not of human health concern. Furthermore, there is potential that the vector itself may induce immunity against ERD acting as a bivalent vaccine. Previous reports using attenuated bacteria as a vector for gene delivery indicate that release of pDNA is enhanced by death of the bacteria inside the host cell [17]. One strategy to achieve intracellular rupture of the vector is through the use of cell wall deficient bacteria. In this proposal, a strain Y. ruckeri lacking the ability to synthesize the cell wall component diaminopimelic acid (DAP) will be exploited. In the absence of an exogenous source of DAP, the bacteria cannot synthesize the peptide cross bridges of the peptidoglycan cell wall and will undergo autolysis during subsequent growth. Two engineered plasmids will be utilized for the proposed studies, one vaccine and one control. The vaccine plasmid will have the full glycoprotein from Y. ruckeri cloned in downstream of a cytomegalovirus (CMV) promoter. The control plasmid will have firefly luciferase reporter gene also cloned in downstream of a CMV promoter. To summarize these experimental components; bacterial vector strain ÃŽ ±, vaccine ÃŽ ² and control ÃŽ ³ plasmids:      Ã‚   ÃŽ ± Y. ruckeri 11.29Δdap Isolated from Chinook salmon, dapA mutant [18] ÃŽ ²pIHNV-G Complete IHNV glycoprotein (G) gene inserted into pcDNA3 (Fig.1), downstream of CMV promoter [19] ÃŽ ³pLUC Firefly luciferase reported inserted into pcDNA3 (Fig.1), downstream of CMV promoter [18] The proposed model of plasmid mediated vaccine delivery in this system is multi-step: Vector harboring plasmid pIHNV-G enters a cell via endocytosis or phagocytosis Facilitated by Δdap, pDNA is liberated in the cytoplasm via bacterial lysis pDNA is transported to the nucleus Encoded antigen is expressed and processed, inducing an immune response In vitro studies: Transformation of bacterial vector with vaccine and control plasmids Competent Y. ruckeri strain11.29Δdap will be transformed with either pIHNV-G or pLUC by electroporation or chemical methods. Successful transformants will be screened by growth on appropriate media agar plates supplemented with ampicillin and DAP. Examination of transfection frequency via flow cytometry and fluorescence microscopy For tissue culture experiments, Chinook salmon (Oncorhynchus tshawytscha) embryonic cells (CHSE-214), and normal rainbow trout (Oncorhynchus mykiss) gill cells (RTgill-W1) will be employed. These cell lines are relevant to the species and aquaculture context of interest for this research proposal. Both are relatively easy to culture, e.g. not requiring increased [CO2] or temperature. Transformed bacteria (11.29Δdap-pIHNV-G, or 11.29Δdap-pLUC) will be co-incubated with sub-confluent layers of each cell line and transfection frequency / gene delivery with reporter plasmid will be assessed by flow cytometry and fluorescence microscopy. Quantitative assessment of vector invasiveness by gentamicin protection assay Invasiveness of 11.29Δdap, 11.29Δdap-pIHNV-G, and 11.29Δdap-pLUC will be quantitatively assessed by gentamicin protection assays. Briefly, each cell line will be co-incubated with the vector (11.29Δdap-pIHNV-G, or 11.29Δdap-pLUC) at a multiplicity of infection of ~100 bacteria per tissue culture cell and incubated for 2h. At 24 and 48h post-incubation, cell monolayers will be fixed and analyzed via fluorescence microscopy for luciferase expression. In vivo studies: Routine maintenance of rainbow trout treatment / exposure groups in aquaria Transformed bacterial vector will also used to treat O. mykiss in vivo through dosed aquaculture immersion. Adolescent O. mykiss fry will be maintained at 12 °C in appropriately sized aquaria with filtration and aeration and water quality factors (e.g. pH, NH3, Cl) with daily feeding and 5% water changes. In preparation for treatment, fish will be moved to separate isolated 40L aquaria with filtration with feeling and 25% water changes every two days. The proposed experimental treatment / exposure groups will be as follows: 11.29Δdap 11.29Δdap-pIHNV-G 11.29Δdap-pLUC Intramuscular injection of purified pIHNV-G Intramuscular injection of purified pLUC Intramuscular injection of phosphate buffered saline (PBS) à ¯Ã‚ Ã‚ ¸Ãƒ ¯Ã¢â€š ¬Ã‚  vector, plasmid, or injection (anesthesia only) Exposure of O. mykiss with bacterial vector harboring vaccine or control plasmid For treatment with bacterial vector (with or without pDNA), experimental fish will be transferred from their 40L tank to sterile 4L beakers of water (aerated, 12 °C). Appropriate vector will be dosed into the beaker via serological pipette for a final concentration of approximately 107 cfu ml-1, and fish incubated for 1h [18]. Following treatment, fish will be transferred back to their respective 40L tank and maintained as previously described. Exposure of O. mykiss to purified plasmid or PBS control by intramuscular injection For injection treatments, fish will be transported to beakers of sterile 4L beakers of water (aerated, 12 °C) and an anesthetizing dose of Finquel added. Once visibly anesthetized (attenuated movement and muscle tone, reduced respiration) [20], fish will be treated. A volume of 100ÃŽ ¼L of purified pIHNV-G or pLUC resuspended in PBS, will be intramuscularly injected (1ÃŽ ¼g total plasmid), 100 ÃŽ ¼L PBS, or anesthetized with no injection, Once injected, fresh water will be added and fish were monitored until consciousness is regained, and transferred back to their respective 40L tank and maintained as previously described. Lethal challenge of O. mykiss with pathogenic IHNV and quantifying response to vectors After vector or control treatment (14d), fish remaining from each sample group will be challenged with 5104 pfu ml-1 of pathogenic IHNV [21, 22] for 5h. Mortalities will be recorded daily for 30d after viral challenge. At 1, 3, 5, 7 and 14d post vector treatment, or 3, 5, 7, and 14d post injection, fish will be removed from their respective treatment tanks and euthanized with a lethal dose of Finquel. The spleen and kidneys of each fish will be surgically removed, placed in an RNA stabilization reagent, and stored at -20 °C until processing. Organ samples will be homogenized with zirconia/silica beads in a tissue lysis buffer, RNA purified from the homogenate, and synthesis of cDNA performed. Gene expression of Mx-1, Vig-1, TNF-ÃŽ ±1, TNF-ÃŽ ±2, IFN1 and IFN2 [23] will be measured by quantitative polymerase chain reaction (qPCR) relative to housekeeping gene ARP [24], and analyzed by the ΔΔCt method. In previous studies, levels of expression for the genes of interest in thi s proposal have been revealed to be altered in fish exposed to IHNV [19, 23, 25]. Discussion / Impacts: Salmonids, particularly rainbow trout, are globally one of the most scientifically studied and extensively farmed fish [26, 27]. As previously mentioned, aquaculture is utilized worldwide as a major food production method. This necessitates the demand for economically sustainable disease prevention techniques to help preclude economically devastating loss of business due to mortality. This research proposal aims to validate that an attenuated bacterial vector can effectively deliver a plasmid mediated gene based vaccine for IHNV to rainbow trout in vivo, and invoke an immune response that will protect against future exposure to the pathogen. It is postulated that exposure to 11.29Δdap-pIHNV-G will invoke the most significant immune response in treated fish compared to other treatment groups. Furthermore, this treatment will induce the highest level of protection from a subsequent lethal challenge of IHNV. If successful in this regard, an aquaculture based dosing method exploiting plasmid harboring attenuated bacteria would represent a relatively inexpensive and non-labor intensive vaccination method. Further investigating A 16K and 32K cDNA salmonid cDNA microarray have recently been developed and are obtainable through the Genomic Research on Atlantic Salmon Project (GRASP) [28].

Jet Blue Case Analysis Essay

Jetblue set out to provide its customers with a great airlines experience. Neeleman’s goal was to provide customers with â€Å"the types of amenities reserved for the pricier carriers, including wider seats †¦Ã¢â‚¬ ¦and 24 channels of in-flight television† ( Case study pg 400) One of Jetblue and Neeleman’s biggest challenges was to keep offering all these amenities while still competing with the big carriers by keeping their prices 50 to 60 percent lower on the same routes. As they grew and hired more employees they found it harder to maintain the same level of customer service across the board. Also other carriers began to compete with them in the lowprice arena. These bigger airlines had more planes and employees to they were better able to respond to the storm that blanketed New York in 2007. This storm proved to ruin many of Jetblues customer experiences due to the delays and cancellations. Jetblue gave all of their customers refunds and free flights in response to the delays. They were also feeling the effects of the storm longer than their bigger competition since they were understaffed because of pilots being stuck in other states. When the storm hit some flights set on the tarmac for up to ten hours still chancing to be able to leave during the storm. Jetblue could have cancelled these flights earlier and kept customers from having to endure sitting on planes for extended periods of time. If Jetblue had done this then they would have avoided much of the grief they experienced over the next week. Overall Jetblue should have better prepared for the storm by cancelling flights earlier and having extra staff on hand. Whomever was in charge of overall operations should have planned better and is the one who is the most responsible for the lack of preparation. Jet Blue did a great job handling the severe weather in February of 2007. They went above and beyond trying to compensate for the inconvenience and loss of time that their customers endured. They provided $26 million in refunds and vouchers to their passengers stranded in New York. None of the other major airlines offered compensation or even an apology. Even at the companies all time low they did an admirable job offering the JetBlue Experience. Although I commend the way JetBlue handled this difficult situation, there were steps that could have been taken to ease the inconvenience of their passengers. The day before the storm, there were multiple signs of severe weather on the horizon. Snow had already begun to fall and by the early morning the snow became ice pellets and freezing rain. The airline decided to ignore these signs, thus neglecting to warn its passengers of possible delays, resulting in six planes being boarded and ultimately stuck at their gates. Additionally, JetBlue had four incoming planes that should have been directed elsewhere and as a result those planes were unable to reach their terminals because all gates were occupied. If JetBlue would have paid attention to the warning signs and informed their customers, the ten planes and its passengers would have never been stuck at the terminals. The negative consequences JetBlue could face are primarily PR and financial. During the storm in February, the media was constantly covering JetBlue’s â€Å"trapped† customers. Some passengers even went so far as to start a blog called jetblueshostage.com. JetBlue was once known as the leader in service excellence in the airline industry. Now the company is faced with the difficult task of rebuilding its image in the public eye. Directly related to the company’s public image is its stock price. The market lost confidence in JetBlue following the events February of 2007, resulting in the companies stock price falling. In order to get everything back on track the company must first focus on its public image. In order to deal with the unfortunate quagmire the company had found itself in after the snowstorm in the northeast, JetBlue planned to launch a service guarantee known as the â€Å"Customer Bill of Rights† in order to make right what they had wronged. JetBlue announced it would spend $20 to $30 million in effort to appease thousands of stranded customers that were affected. The Bill of Rights works by offering vouchers to customers who experience delayed flights while flying with JetBlue. $25 for flights delayed one to two hours and up to a free round trip ticket for flights delayed up to 6 hours. Will the Customer’s Bill of Rights work in recovering the image JetBlue has tried so hard to create? In my opinion, yes I do think it will. Angry customers who had to deal with the delays on the initial happening will be provided an entire free ticket, and customers who deal with this in the future will be provided with vouchers or tickets as well. What else can an airline company do, errors happen and some may be out of the company’s control. The company must deal with how the error is handled and that is exactly what JetBlue is doing. Several actions and guidelines should be followed by JetBlue in order to insure the companies viability and future success. The launch of the Cutover’s Bill or Rights was a good step in the right direction, but company executives must work closely with their public affairs team to raise its awareness. JetBlue executives must also work with marketing executives to promote the Customer Bill of Rights with large stakeholder groups and already existing customers. JetBlue executives must support this bill of rights 100% in order to restore the company’s image. This means following their promise and actually providing vouchers for every single delayed flight. Customer Bill of Rights should also be leveraged as an advantage in comparison to its competitors. Considering JetBlue was the first to implement such a thing, advertising it as an advantage my pull customers in and keep current customers.

Food for Thought Essay

The well known expression, you are what you eat, is even more true as we get on in years. If we had eaten the food that was good for us in our younger years, the chances of staying healthy longer will improve as we get older. Also, the likelihood of maintaining a high quality of life throughout our senior years increases. Reading nutrition columns in newspapers and magazines or from other media sources is a good way to keep updated of current food and health related discoveries. How can we be able to estimate and gauge the truthfulness of scientific studies about food? Linda Kulman gives us good advice about how to do just that in her article, Food News Can Get You Dizzy, So Know What to Swallow. I believe that for a person to be able to make healthy dietary choices a person needs to be educated as to the credibility of healthy dietary options. Primarily, to achieve and maintain good health, food from all the major food groups should be eaten in proper proportion and regularly. Therefore, no one food is able to maintain good health when eaten alone. For instance, â€Å"No foods are so good that if you ate them to the exclusion of all else, you would be healthy,† says M. R. C. Greenwood, a biologist and chancellor of the University of California-Santa Cruz (Kulman, 2012, p. 141). Making the correct dietary choices was, and continues to be a difficult one. Confusion can turn to frustration when many reports and studies contradict each others findings. Canada’s Food Guide to Healthy Eating will give us the basics on what constitutes a healthy diet. The food guide basically recommends to eat in moderation and to eat a large variety of foods. The flip-flops of nutritional recommendations by the scientific community are causing bewilderment with many people who are trying to achieve and maintain a good healthy diet. Furthermore, the tale of fiber and its claimed shielding effect against colon cancer show how uncertain science can lead to confusion. Fiber helps food go through the digestive track faster, reducing the time carcinogens make contact with your intestinal walls. Studies of high fiber eating population and experiments with mice and rats resulted in giving the fiber hypothesis some credibility. Even though the evidence for higher fiber consumption reducing cancer risk remained uncertain, in 1984 the American Cancer Society made its first specific recommendation to eat fiber to help prevent colon cancer. Researchers with the Nurses Health Study in Boston tracked the diets and health of more than 88,000 American female nurses since 1980 and found that nurses who ate about 30 grams of fiber a day got colorectal cancer just as often as the average American, who consumes just 13 grams (Kulman, 2012, p. 143). Two additional study results showed that eating more fiber does not reduce the risk of getting colon cancer. These studies show that there was no significant difference of colon cancer risk between man and women. Researchers continue to suspect that whole fruits and vegetables as well as whole grains are protective against colon cancer. In conclusion, the fiber story is an example of how reporting results of scientific food studies while the studies are incomplete, can lead to many people becoming discouraged from believing future reported food study findings. Usually the cause of such distortions is the incompatibility between the needs of science and those of the News Media. â€Å"The way a lab finding makes its way to the headlines is like a conveyor belt,† explains the Statistical Assessment Service’s Murray. â€Å"At each step there is a potential distortion. Where science is contingent and unfinished, journalists want something definitive (Kulman, 2012, p. 143). The most frequent complaints about news reports is that they tend to leave out information that would help readers decide how seriously to take a new finding. The News Media should not be the only one to take the blame for the reporting of incorrect information about study findings. Scientist can get very enthusiastic when reporting their findings to the News Media and can easily be misinterpreted as to the significance of their findings. Scientists are often motivated to embellish their claims to get greater attention and more research funding. This is an unfortunately situation for those of us attempting to make an informed choice for a healthy diet.

Memorial Day The Women Behind Its Origins and History

While Veterans Day in November is to honor all those who served their nation in war, Memorial Day is primarily to honor those who died in military service. This all-American holiday has its roots in unexpected places. Commander in Chief John A. Logan of the Grand Army of the Republic issued the 1868 proclamation declaring the first Decoration Day, which was celebrated with a large memorial observance at Arlington National Cemetery, with about five thousand attending.  Those attending placed small flags on the graves of veterans. General Ulysses S. Grant and his wife presided at the ceremony. Logan credited his wife, Mary Logan, with the suggestion for the commemoration. The role of his wife may explain why Grants wife co-presided over the ceremony. But the idea had other roots, as well, going back at least to 1864. A First Memorial Day In 1865, a group of 10,000 freed slaves in South Carolina  along with a few white supporters—teachers and missionaries—marched in honor of Union soldiers, some of whom had been Confederate prisoners, reburied by the freed black Charlestonians. The prisoners  had been buried in a mass grave when they died at the prison. While this ceremony can be called the first Memorial Day, it wasnt repeated, and was soon nearly forgotten. More Direct Root of Todays Celebration The acknowledged and more direct  root of Decoration Day  was the practice of women of decorating the graves of their loved ones who had died in the Civil War. Memorial Day was celebrated on May 30 after 1868.  Then in 1971 the celebration was moved to the last Monday in May, to make a long weekend, though a few states kept to the May 30 date. Decorating Graves In addition to the Charleston march and a long practice of both Union and Confederate supporters decorating the graves of their own, a particular event seems to have been a key inspiration.  On April 25, 1866, in Columbus, Mississippi, a womens group, the Ladies Memorial Association, decorated the graves of both Union and Confederate soldiers. In a nation trying to find a way to move on after a war that split the country, states, communities and even families, this gesture was welcomed as a way to lay the past to rest while honoring those who had fought on either side. The first formal observance seems to have been on May 5, 1866, in Waterloo, New York. President Lyndon Johnson recognized Waterloo as the Birthplace of Memorial Day. On May 30, 1870, General Logan gave an address in honor of the new commemorative holiday. In it he said: This Memorial Day, on which we decorate their graves with the tokens of love and affection, is no idle ceremony with us, to pass away an hour; but it brings back to our minds in all their vividness the fearful conflicts of that terrible war in which they fell as victims.... Let us, then, all unite in the solemn feelings of the hour, and tender with our flowers the warmest sympathies of our souls! Let us revive our patriotism and love of country by this act, and strengthen our loyalty by the example of the noble dead around us.... By the late 19th century, with the rise of the Lost Cause ideology in the South, the South was celebrating Confederate Memorial Day.  This separation largely died out in the 20th century, especially with the change in name of the Northern form of the holiday from Decoration Day to Memorial Day, and then the creation of a special Monday holiday for Memorial Day in 1968. Some veterans groups were opposed to the date change to a Monday, arguing that it undermined the real meaning of Memorial Day. Other cities which claim to have been the origin of Decoration Day include Carbondale, Illinois (home of General Logan during the war), Richmond, Virginia, and Macon, Georgia. Official Birthplace Declared Despite the other claims, Waterloo, New York, got the title of birthplace of Memorial Day for a May 5, 1966, ceremony for local veterans.  Congress and President Lyndon B. Johnson issued the declaration. Poppies for Memorial Day The poem In Flanders Fields commemorated fallen war dead. And it includes a reference to poppies.   But it was not until 1915 that a woman, Moina Michael, wrote her own poem about cherishing the Poppy red, and began encouraging people to wear red poppies for Memorial Day, wearing one herself. Moina Michael is featured on a 3 cent postage stamp in the United States, issued in 1948.