Identification of a Meat Sample DNA Technique using Polymerase Chain Reaction

Many DNA based diagnostic tests have been developed after the discovery of structure, function and duplication were discovered. Over the last 5 decades these techniques have undergone rapid changes and modifications to suit different applications such in phylogenetic relationships and evolutionary tree constructions of different taxa including all the varieties of living organisms. The techniques have been created for extracting gene sequences of known disease related and desirable traits in all organisms. These gene genes have suitable modified for their stable transfer into other taxa, integration, and later, detection of the same by many diagnostic techniques in the host recipient taxa. One of the most popular techniques has been based on the Polymerase Chain Reaction, a property of a DNA polymer formation by an heat tolerant enzyme called the Taq polymerase from thermo-stable Thermus aquaticus ( a fungus). In this paper this reaction has been used for the detection of a meat sample of an unknown organism. Its many applications have been briefly described. 
Identification of unknown meat samples has been very crucial in various kinds of situations such as food industry. In food industry meats of many organisms look alike with minor differences in texture, color, softness. Meat from cheaper and easily available organisms can be adulterated with processed food. Meat can also be contaminated with harmful pathogenic bacterial andfungal organisms andor also their toxins.

    Since the discovery of the molecular structure of DNA in 1953 by Watson and Crick and later a several researches on sequences, functions and expressions, many new areas of applications of DNA have become easier. No other diagnostic test has been found to be as real and true as the DNA based tests. DNA characterization tests have been of major value in evolutionary understanding and phylogenetic affinities of any organism. 

       Among the DNA tests many versions have been developed to achieve precision, economy of time and costs and minimizing the use of hazardous chemicals such as radioactive and highly toxic substances. In most of them, restrictions enzymes have been of crucial and key role. A restriction enzyme cuts the DNA at very enzyme specific sites. These DNA fragments are subjected to separation by electrophoretic methods on agar or agarose gels. The separated DNA fragments are probed with known radioactively labeled DNA sequences. These probes bind to the fragments which have these very sequences partially or fully. The technique has bee simplified considerable for diagnostics in shorter time by the polymerase chain reaction methods.

    A rapid test for meat from unknown animal sources has been described many times. Many tests have also been described and published. Buntzer et al (2005) used the satellite DNA markers as probes. Earlier Locksley and Beardsley (2000) described a DNA based method for authentication of the source. This has many applications in the food industry 

In this paper a molecular analysis experiment was done to characterize a meat tissue of unknown animal origin. DNA was extracted and mitochondria specific DNA (mtDNA) fragments were amplified by polymerase chain reaction. The positively amplified sequences were then cycle sequenced using a non-radioactive fluorescent dye. The DNA sequences so obtained were found to be 293 base pairs. These sequences were then compared with the large vertebrate DNA sequence Data Base on computers. The comparison provided the most probable identity of the organisms involved.

Materials and Methods
DNA Extraction  DNA from the unknown sample meat tissue supplied was extracted using the Qiagen animal DNA extraction kit ( 69506). The meat sample tissue was thawed in a water bath set to 55 0 C.  The tissue held by a pair of sterile was cut into A small piece (about 25 mg) was tissue was cut out by a pair of sterile scissors  from the thawed tissue held by a pair of sterile forceps. This piece was dispensed into a 1.5 ml tube labeled with the supplied meat tissue sample no. on the lid of the tube by a sterile tooth pick. Into this sample 180 ul of ATL buffer using a sterile tip on a blue pippettor. The tissue was lysed using 20 ug20 mg per ml Proteinase K using a yellow pippettor and mixed thoroughly. This tube was then incubated at 55 0 C for 45 minutes while shaking.

A water bath was heated to reach 70 0 C and allowed to remain at that temperature. The lid of the sterile DNAeasy spin column was labeled with the provided meat sample number. This was into a sterile 2 ml collection tube. Another 1.5 ml sterile tube was labeled with the sample and T DNA (Total DNA). After thorough mixing the sample, the lysis was stopped using 200 ul AL buffer. After mixing this 200 ul of 100 ethanol was added and mixed again. This mixture was transferred with a pippettor into the spin column in the 2 ml labeled tube. This was centrifuged at 8,000 rpm.  After slowly decanting the supernatant, the spin column was put back into the collection tube. The precipitate was washed with 500 ul AW1 buffer and spun again at 8,000 rpm. The spin column was put again into collection tube washed again with AW2 buffer and then spun at 11,000 rpm for 3 minutes. After decanting the supernatant the DNA easy spin column was taken again into 1. 5 ml tube. To elute 200 ul AE buffer was added to the membrane to the spin column. This was left for 1 minute at room temperature. This was spun at 8,000 rpm and then the spin column was removed into dust bin leaving DNA in the collection tube. This DNA sample was then electrophoresed to determine the success of the above procedure.
Polymerase Chain reaction.- To each tube 47 ul of supplied Master Mix (containing 35.5 ul ddH2O, 10 ul of salt buffer including Mg Cl2, 10 ul of dNTPs, 0.5 ul of Taq polymerase) with separate new pippette tips. The to each the following were added using a new and separate pippette tips
1 ul of forward primer (NM)
1 ul of reverse primer (PIL)
1 ul of our test DNA as prepared above

        The lids were tightly closed and put on a strip tube each.
    Thermal cycling was conducted as guided with a GENE AMPR PCR System 9700. This goes through40 cycles of 1 min each (94 0 C for 3 minutes for denaturation1 min at 54 0 C for primer annealing and 4 min at 72 0 C for primer extension) after initial incubation at 94 0 C for 3 minutes. There was a final step of 10 minutes at 72 0 C for termination of the Polymerase Chain Reaction (PCR).
    This DNA sample was electrophoresed on 1 agarose gels. These gels were stained with ethidium bromide to reveal the ethidium bromide stained bands under UV light.
Cycle Sequencing Reaction
    Quantity of clean up PCR product was determined by comparing the intensity of ethidium stained bands of the test meat DNA sample with that of the PGEM band. If the intensity was equal 2 ul of the test meat DNA sample was used. In our sample preparation intensity was more than the intensity of the Ethidium bromide staining PGEM band. Therefore 1 ul of the sample was used.
To the 0.2 ul tube labeled as meat sample number, the following solutions were added
19 ul of dd H2O
1 ul of Primer ND4
1 ul of PCR test sample DNA product
3 ul of Quick Start DTCS (Direct Terminator Sequence mix) from Beckman Coulter Inc.)
The tubes were tightly closed for thermal cycling reaction. Gene Amp PCR System 9700 was used for thermal cycling. This goes through 40 cycles  20 seconds at 96 0 C for denaturation, 20 seconds at 50 0 C for primer annealing and 4 minutes at 60 0 C for primer extension.

Cycle Sequencing Reaction Clean up 
    The meat test sample DNA obtained as above is once again is cleaned. To the tube 5. 0 ul of Prep solution (2. 0 ul 100mM EDATA, 2.0 ul of 3 M NaOAc, 1. 0 ul of glycogen) is added. To this 60 ul of cold 95 ethanol is added. This was mixed thoroughly and centrifuged at 13, 200 rpm for 15 minutes.  The supernatant was removed by a pipette without disturbing the pellet.  Then 200 ul of cold 70 ethanol was added, mixed thoroughly, spun again the supernatant was once again removed. Cold ethanol 70 was added again and centrifuged at 13, 200 rpm for 5 minutes. The sample was air dried for about 30 minutes at room temperature till all the ethanol was evaporated. 
Electropherograms, Sequence Analysis
    The success of the above reactions was assessed by electrophoresis in 6
Polyacrylamide gels in the Beckman Coulter CEQ 8800 DNA Sequencer. This contains the LASER which reads the signals from the fluorescently labeled ddNTPs    
The DNA sequence used for comparing the 16 base pair ATCGATCTACGATCG sequence.
The sequence of the test meat sample as obtained in the Beckman Coulter Sequencer was compared with the publicly available annotated  sequences 13 billion bases from over 100,000 available at the NCBI ( National Center for Biotechnology Information of the National Library of Medicine - National Institutes of Health, USA  web site). This Gene Bank of this Center contains other world wide updated information on Gene Bank,  Blast, Taxonomy and published research papers from a large number of world wide sources.
Basic Local Alignment Search Tool (BLAST) is used for carrying out the alignment and similarity index analysis.

     Table 1 gives the list of animals with Similarity Index over 75. Maximum ID observed was in the range of 75  81. The logical choice of the animal closest to the test meat sample may most probably be Drymarchon corais which showed the maximum similarity of 81.

One of the NCBI responsibilities is to facilitate the easy methods for data searches in addition to creating vast data bases on molecular biology information from all the published and available real data and storing them in manageable manner.   In searching for similarity information a parameter of low E value is chosen. In our experiment an E value fixed was 0.0  e 0.154  6e 0.21  1e 0.13  5e 0.07.
Nine plant species were found to have the range of high similarity index between 74 - 81. Among them Drymarchon corais was found to be the closest with 81 similarity to the meat test sample.
Many varieties of DNA based tests have been described and published. All these publications have facilitated the use of DNA based tests for various purposes such as the authentication of the source of meat Bunter et al., 2005 identification of meats from different samples (Locksley and Beardsley, 2000), evaluation of pathogenic Data et al.,  for Hemolytic  Listeria monocytogenes) and infectious organisms (Ellingson et al., Salmonella Dragan and Avraham, 2000 Salmonella in poultry, Fluit et al., 1993) and or their toxins causing serious health hazards to consuming humans or cattle and poultry (by colony hybridization, Datta et al 1987).
The attraction of the newly developing DNA technology for detection of large numbers of sample in short time was reported as 50 samples in just 4 hours (Buntzer et al 1995). Polymerase Chain reaction was used for pork in heated in meat products (Meyer et al., 1994) 


As a child goes through the facts of marine life, it is also an ongoing imagination of a place where reality and unknown meets. Thus, it would be better to reinforce the child with not only facts but also images that captures the portion of reality underwater. This could not be possible without the advent of underwater photography. Truly, underwater photography paved the way for scientists and common people alike to understand and admire the marine world in a deeper extent. The pictures taken by the underwater photographers helped marine biologists by giving them the exact pictures of the species that are yet to be classified and it also reflected the natural habitat of these animals which needs conservation. Through these pictures, they were able to study further the physical attributes of each species. Also, with the help of these pictures, new found species are easily identified and classified. Underwater photographs also opened the minds of the people to the importance of the natural resources found underwater because they can visualize the exact habitat of these animals and they can also appreciate the unique beauty of the underwater world. Thus, it leads to more determined actions in the preservation of this wealth.

Contrary to popular belief, the worlds first underwater photograph was not taken by Emile Boutan but it was taken half a century earlier by an Englishman, William Thompson as clarified by Alary and Gilbert in their article entitled Photo 101 Part 1 of underwater photography history and prerequisites. As shortly discussed by Nick Baker in his article William Thompson  The Worlds First Underwater Photographer, William Thompson was a wealthy and intelligent Gentleman from Weymouth in Dorset. He was married to Sarah Slade and moved to the house near the seashore. This intensified Thompsons interest in the marine life. He became an expert in anemones and even discovered several new species of seaweeds. He was also an acknowledged expert in molluscs and had many important ornithological observations. 

The first ever underwater photograph was taken using a wet plate collodion as itemized by Nick Baker in William Thompson  The Worlds First Underwater Photographer. To further understand the technique that was used and appreciate the primitive way the photo was taken, it is better to discuss first on the technicalities of the process.

    As discussed by Baker in William Thompson  The Worlds First Underwater Photographer, collodion process was revolutionized by Frederick Scott Archer. This process utilizes a collodion, a viscous liquid made from a dissolved gun-cotton in alcohol and ether. This process was perfect for binding light-sensitive chemicals onto a glass plate and this has a reduced exposure time as compared to other primitive techniques at that time. The decrease exposure time was of great advantage at that time however, one main disadvantage of this process is the preparation of the materials and the process itself. The photographer has to prepare his plate first by spreading onto the glass a fresh collodion. He then has to synthesize it and mount it in the camera. After taking the picture, the plate will then be retrieved and the picture developed and fixed and all this time, the glass should remain wet. Thus, the photographer, may he be professional or amateur, should be familiar with the chemicals he is using. This also means that the photographer should carry all his equipments, including a darkroom tent, to the location of the photography, which was a great inconvenience in the part of the photographer. However, this was not a problem with William Thompson since he was driven by his enthusiasm of photography and even attempted new feats in underwater photography. The image captured by Thompson was an ultimate disappointment but it cant be denied that the image was an evidence of a technical milestone.

    After several years, a photographer from National Geographic Magazine and William Harding Longley produced the first ever colored photograph taken underwater as documented by the National Geographic Magazine First Underwater Color Photos. It was the interest of William Longley that started it all. He wanted to photograph the fishes he was studying in their natural habitat in full color. However, the technology at that time was still deficient for this kind of notion. He first tried autochrome glass plate but this only permitted him to take pictures of still objects like anemones and corals. To add up on the limitation of this technology, it needs 10-12 seconds exposure time for anything to register in the plate and the pictures were still disappointing. His interest unites with the interests of the President and Editor of National Geographic. So he was accompanied by a knowledgeable photographer from National Geographic and improvised the autochrome technique by creating a solution that coats the plates and cuts the exposure to significantly. Nevertheless, exposure time was not the only problem they have to overcome the blue hue of the sea. Thus, they attempted a very dangerous stunt. They used a magnesium flash powder which when ignited explodes and gives of a very bright light that was equivalent to 2,400 flash bulbs. The tedious and dangerous task was proven to be successful. For the first time, a full color of underwater photo was published, which was published in the National Geographic magazine in their article First Underwater Color Photos.

    Sad to say, nobody in the photography or marine biology world repeated the experiment as noted in the National Geographic Magazine in First Underwater Color Photos. The pictures remained only a lonely monument. On the other hand, their success was well treasured by the National Geographic and became the great tradition of National Geographic underwater achievements. After this another milestone in photography, decades was had to be spent to experience the convenience and the success of fast-color film.

    Owing to the technical advancements which occurred there after, underwater photography has now developed a technique that captures the fluorescence of corals. Fluorescence of corals is truly a breathtaking view but at first, it didnt receive much attention from marine biologists. Thanks to the efforts of some biologist, like Charles Mazel who intently studied this characteristic of corals. He did not only study the importance of fluorescence of corals but he also developed a technique that allows underwater photographers to shoot even under ambient light (Daubilet 32).

    Photographers use different kind of light to elicit the corals ability to fluorescence. Unlike the usual white light used in photography, in underwater photography, to elicit the fluorescence capability, ultraviolet light should be used (Daubilet 37).

As Charles Mazel explained it in the article Charles Mazel on Coral Fluorescence, he differentiated the fluorescence phenomena in physics and biology. In physics, this process occurs when one color in the white light is absorbed in a substance and it is then transformed into another color. However, this is not applicable in biology. Fluorescence originates from few sources in corals. It comes from the intense colors of green, orange and yellow which are present in the fluorescent protein in the coral tissue. For example, a deep red fluorescence is observed in algae. This red fluorescence came from the chlorophyll content of the algae. On the other hand, orange fluorescence is exhibited from phycoerythrin, which is a pigment found in red algae and cyanobacteria which has a specific function in photosynthesis. Fluorescence is not only present in corals and algae. It can also be found in other reef fauna, like shrimp, crabs, fish and more. Although fluorescence is usually appreciated among corals, not all corals exhibit this phenomenon. Thus, one may wonder the significance of fluorescence phenomenon.

    There are several theories attempting to explain fluorescence of these animals however, these are yet to be proven. However, scientists discovered that the fluorescence of juvenile corals helps them identify newly settled young corals in the same reef. This is significant information because it aids scientists or marine biologists in their study of coral recruitment. This was explained by Charles Mazel in Charles Mazel on Coral Fluorescence. He also added that fluorescence of corals doesnt only help marine biologists understand underwater life better it also helps researchers find medical significance of corals. The genes that code for the fluorescence ability of corals are inserted to cells of different laboratory subjects. This results to fluorescence of a specific part of the subject. Thus, it can be used in monitoring the developing organs or even exhibit the effectiveness of anti-tumor treatments.

    Through underwater photography, one can truly say that technology has greatly contributed in the understanding of once an unknown and mysterious world. Through the efforts of photographers, scientists, and marine biologist, the marine life was gradually uncovered and explored even by common people. It did not only advanced by itself by improving the technology in photography but it also expounded our knowledge of marine life through its course.


In recent times, there has been an increase in awareness as regards to the meaning and the effects of air pollution on our environment. This might be as a result of education and peoples perception of their health need. Air pollution comes in different forms and it affects humans, animals and plants in varying degree. An important form of air pollution in our environment is the acid rain. This form of air pollution is now a serious environmental issue in developed countries of world especially in Europe (Likens, Driscoll  Buso 1996). Acid rain is a term that has been used to describe a form of air pollution in which airborne substances which are produced from industrial waste or electric utility plants are released into the atmosphere. These substances undergo chemical changes and result into the formation of acidic compounds. Acid rain also denotes deposition of acidic compounds into rain, snow and fog hence could be referred to as a form of precipitation (acid precipitation).
Acidic substances are found to be widely distributed in nature. They are found in our stomach, and in fruits such as grape and vinegars. They can also be produce in the laboratory by chemical reactions. Base which when mixed with acid neutralizes the effects of the acid also occur in diverse forms. It can be produce synthetically in form of ammonia or occur as a form which is found as a constituent of pancreatic juice in humans. Some medication such as antacid also contains bases.
Proper understanding of the acid such as it common sources in the environment and how its being measured are essential to the assessment of its impacts on our environment. Acids describe corrosive property of a substance and a substance is said to be acidic if it can release hydrogen ion to form a solution. Such substance acidity could only be assessed by its pH value. PH is a scale that is used to determine the acidity or alkalinity of a substance and it range from 1-14 with a neutral point being placed at 7. Any substance or compound greater than 7 is said to be basic in nature while those less than 7 are said to be acidic in nature.
The term acid rain has been used to describe a rain with a pH value below 5.6 and has been found to be much more common in highly industrialized communities. The main contributing compounds to this form of air pollution are oxides of sulphur and nitrogen which are released into the atmosphere and then get converted into substance such as sulphates and nitrates which in turn yield sulphuric acid and nitric acid in the presence of moisture. Substances that are released into the air are the main contributor to acid rain. These substances which are oxides of sulphur and nitrogen are majorly from exhaust fumes of vehicles, industrial plants and power generators. Increase in human use of all these forms of machines can be attributed to increase release of fumes or all the poisonous gases into the atmosphere and hence increasing the level of formation of acidic compounds in the rain.
Apart from industrial release of these poisonous substances, minor amount can also be contributed by burning of fossil fuels and various forms of agricultural activities. All these sources go a long way in contributing to the increase incidence of acid rain in various communities. Nature has a way of maintaining a state of equilibrium. This can be seen in our environment in the sense that some naturally occurring bases found in the air and soil or those release to the air probably as a result of decay of dead animal have a way of neutralizing the corrosive nature of acid that has been released into the atmosphere or those found in the biosphere.
As was stated in the introductory paragraph, the increase in awareness as regards to deleterious effects of this form of air pollution, has led to formation of several policies and strategies against release of substance that lead to acid rain by government so as to reduce the level of acid rain hence reducing it effect. Through good policies and strategy when formulated and properly implemented it can actually help reduce the effects of acid rain.
Acid rain has several negative effects on plants, animals, soil, surface water and other human made structures. The effect of acid rain on soil is usually attributed to the dissolution of the important soil nutrient and washing them away hence slowing down their growth (Johnson, Driscoll  Eaton 1981). It has been associated with a diminishing fish population and malformation hence causing a potential human effect. This has strongly influence the perception that there are both indirect and direct effects of acid rain on human population.
Analysis of the effect on acid on fresh water ecosystems have shown that acid vulnerable regions are much more numerous and widespread contradicting what was believed in few years back. In this analysis, lakes and streams were found to be more vulnerable to acidification by the effects of acid rain and this are in the region of North America where their lakes have lost the ability to neutralize the acid hence resulting in increase rate of biological damage. Many invertebrates were found to be very sensitive to all these changes with host of them disappearing at acidic pH low as 6.0. Some areas have also shown a decrease in the rate of acidification attributed to consequence of acid rain are on the decline, this was attributed to a recent decreases in sulfur oxide emission (Schindler 1988).
There is high level of acid rain in the United States with the average annual pH of 4.05-4.3 where the contribution of sulphuric acid has been found to account for about 55-75 percent of measurable acidity. Clean Air Act which was passed in 1990 made an impact in the reduction of sulphur dioxide being released from the school. However, this has not significantly precluded forest and aquatic ecosystems so as for such habitat to recover from the effects of sulphuric acid (Likens, Driscoll  Buso 1996).
    The introduction of the Clean Air Act of 1990 has been attributed to current reduction of acid rain being experienced in certain parts of United States especially the Eastern States. This region of the country has long being recognized has the region with highest occurrence of acid rain the united states and the pollutant founds to largely contributes to this form of air pollution are those from local sources and sources located from the Midwest. Out of all the implicated sources of acid rain, 30 percent nitrogen oxides are released from the power generating plants, 30 percents are attributed to those released from automobiles while additional 25-30 percent are from agricultural sectors. Other minor sources are those nitrogen oxides that are released from the process of lightening and other biological process. (Penn State 1998).
The concentration of sulphate ion that are found to be implicated in acid rain was also found to be on the decline in the Eastern part of United states from the period of 1995 through 1997 as compared those analysis done before that period. This shows that there is actual reduction in the level of acid rain in that community but the concentration of nitrate ion which also contribute to acid rain were found not be on the decline hence showing that the clean air act of 1990 that was formulate to reduce acid rain occurrence were limited in it effectiveness in that it only help reduce those emission associated with the release of sulphur dioxide (Lynch, Bowersox  Grimm 2000).
Acid rain leads to various forms of destruction affecting both the living and non living things. This is because it is corrosive to metal, stones and paints which are non living things while it is poisonous to living things ranging from aquatic organisms to terrestrial ones. It affects the growth of plants, cause damages to leaves hence reducing yield. It effects on the plants can be measured in the laboratory by simulating the acid rain. This is usually done by conducting an experiment that allows the scientist to grow certain plant in a controlled group and experimental group simultaneously. Effects of acid can then be measure when the scientist ensures that all other variables are made constant for both the control and the experimental group. Features of plant growth can then be evaluated after certain period of time.
    Since acid rain has been found to be directly destructive to both plant structure and indirectly destructive to the growth, it effects on the growth and leaves of the sunflower will be evaluated in the experiment. Two hypotheses were tested
1) Ho there is no observable difference in the growth of sunflower samples obtained from a control environment when compared to samples grown from the experimental environment.
2) Ha there is an observable difference in the most probable number of fronds in samples obtained from a control environment when compared to samples taken from the experimental environment.
The Null Hypothesis will be tested.
Method and materials
2 peat cup
Plant seed
pH meter
Ruler or tape rule
Cup of vinegar
Two peat cups were collected (one for the control and the other for experimental group. Each cup was filled with potting soil and a hole of 4 cm deep was made in the soil. Three sunflower seed were placed in each hole that has been created in the soil and the seed were then covered with soil. The two cups were watered until the seedlings started sprouting. In the control group, 50 ml of water is used to water the seed. In the experimental group, 50ml of a mixture of vinegar and water was used to add water to the seedlings in the experimental group. The two cups that has been watered were then place at an indoor location.
 The two pots after placement at the indoor location were then left for some weeks so as to assess the growth. The heights of the plant, the total number of leaves and the pH of the soil in each cup were measured at least three times a week. Subsequently, 20 ml of water are being added to control group everyday while the same volume but made up of mixture of vinegar and water is used to water the experimental group. This process of watering the two groups was then continued for the period of four weeks so as to be able to evaluate the effect of lowered pH on the growth of the seed.
It is also very important to note that while the experiment was being conducted, all other variable apart from those of important to the experiment were made to be constant. Variables such as the size of the cup, the amount of time for the experiment, the nature and amount of soil, the volume of liquid and the amount of seed that could directly or indirectly influence the growth of the plant were ensured to be constant.
ControlDateHeight of LeavespHSignature111720093cm26111920098.526112020099.926Ming Lin112520091336Prof. Wildt1129200916.636Ryan Moran113020091746Qian Gu1201200917.54.56Ryan MoranExperimentDateHeight of LeavespHSignature111720092.5cm26111920096.25cm26112020097.5cm26.5Ming Lin1125200910cm25.5Prof. Wildt1129200910.5cm25Ryan Moran1130200911cm24Qian Gu1201200915.5cm34Ryan MoranConclusion
In this experiment, the result shows that at constant pH, there is a constant growth of the sunflower in terms of height and number of leaves. When the measurement were left for certain number of days without measurement (that is about 5 days) appreciable growth in terms of height were noticed showing that the sunflower is growing at a specific rate. In the experiment group, the height of the sunflower was comparably less when compared to that of controlled group at the same pH. This might have been a result of lowered amount of water since the volume of water in this case is about half of that used in the controlled group. When the pH was increased, there is no significant improvement in growth when compared to the controlled group. When the pH of the soil fell below 5.5, the noticeable or significant impairment seen is in the number of leaves which was found not to increase when compared with the controlled group with the a constant pH. These changes help reject the null hypothesis which indicate that there is no observable difference in the growth of sunflower samples obtained from a control environment when compared to samples grown from the experimental environment hence supporting the main hypothesis. This results shows that lowered pH had an impact on the growth of sunflower in terms of number of leaves and the height of the plant.
     The question that needed to be reviewed or subjected to further research is that why is it that when the pH of the experimental group was slightly increased, there is no appreciable changes. Another major factor that needed to be considered was the type of acid in the vinegar that was used. It might be other forms of acid that has little effects on the growth of sunflower. The initial pH of the soil was not also analyzed and this might have had some effects such as neutralization of the acid added to the soil. Because a soil with high alkalinity will show tendency of neutralization of any form acid added to the soil.
My recommendations are that the government should formulate new strategies and Acts that will help reduce the release of emission especially those associated with oxides of nitrogen so as to reduce the impact of acid rain in our environment.

Genetic Transformation of Bacteria with pGLO

Genetic transformation is an application of numerous molecular biology researches leading to very basic structure and function of DNA in all the living organisms. A thoughtful assembly of information generated in knowing various other molecules and their relations with DNA structure and function including reproduction of DNA itself has been put into many meaningful applications. These include indirect (molecular mediated) andor directed therapies for several major and important human diseases. Essentially this involves the basic genome sequence of any organism partly or fully and locate the undesirable sequences and correct them through genetic engineering methods. A major outcome of this is also the genetic transformation of many important and major crops of the world such as maize, rice, sorghum and others. There are many crops which are in the pipeline for total genome sequence. But even before that the techniques have been used efficiently for real genetic transformation of many crops and higher plants with several gene sequences for many qualitative and quantitative traits such as productivity, pest and disease resistance and their better tolerances to even abiotic stresses.

    Preparation of gene sequences for inserting them into a crop requires some parameters such as marker gene sequence. A marker gene sequence is one which can be detected at nearly every step of the transformation process. The detection process may be just physically and also for their actual expression. One of the most popular marke gene has been the gene for an antibiotic (e.g., ampicillin) resistance. Researches have been done to simplify and shorten the sequence of the process of genetic transformation for accuracy and predictability and efficiency. Transformation process has been simplified to such an extent that even such tests for structural or functional abilities of the gene to be transferred can be simply vies at the bacterial culture. A recent example is the Green Fluorescence Protein (GFP) gene extracted from genome of mutated jelly fish, Aequoria Victoria. The GFP mutant has the ability to survive and display enhanced bright green fluorescence, hence the name GFP. To the pure functionally competent sequence of this gene, another previously established marker gene for ampicillin gene has been attached. This compound gene has been effectively used for demonstration of the bacterial genetic transformation. This gene tagged with ampicillin resistance gene was obtained from a commercial supplier for bacterial transformation of E. coli HB101 K12. As publicized by the supplier and guided according t the suppliers instructions for its use except the step of using heat block at 42 0 C for 50 seconds (as a substitute for the use of water bath), there was no growth of any bacterial colonies.

This post discusses the various plausible reasons for any repeated trials in the light of published information on the failures of the bacterial transformation process.

Genetic transformation is an accepted term for the use of molecular biology and genetic tools for altering an organism at the molecular level. Genetic transformation is a reality today. Virtually nearly any organism can be transformed genetically with a desirable disease curing, preventive and or resistant gene. The only prior requirement is that the DNA sequence of the desired should be extracted or constructed appropriately to suit the final objectives and should be easily transferable to the desirable recipient host species. This should be stable integrated in the host genome and should be made to express and function normally in the environment of the new genome of the recipient.

The present state of this knowledge is based in numerous researches carried out by Hayes, 1961 and several others and tons of published information is available on the methods, protocols, techniques, and their possible applications in molecular genetic improvement of animals, crops, beneficial microbes (e.g., Kosuge et al., 1983). Genetically transformed microbes have proven very valuable in large scale industrial production of insulin, many healthcare biomolecules, peptides, new generation antibiotics, and many more. They are potential in treating even the effluent areas as in the production of bioethanol as a renewable source of energy.

Basically deals with proven knowledge that purified DNA segments can enter into cells and get integrated into the nuclear material of the cells in such a manner that the new introduced fragment can even start functioning with normal expression. Thousands of DNA sequences have been characterized for their structure, location in the total nuclear DNA and their function. Now even complete genome sequences are established for man, major crops, Caenorhabditis elegans, yeasts and many bacteria. Such sequences have far reaching potentially useful implications in therapy of human diseases and infections, crop diseases and pests, and animals.

For all of these kinds of possible applications and implications many more directions of researches have produced results which gave been synthesized to realistically develop such workable schemes with nearly mathematical precision. A DNA sequence characterized is prepared and made ready for introduction into the host organism. A well known DNA sequence established for ability of gene promoting activity (called promoters) is attached. The so joined sequence of two different gene sequences is then attached to another gene sequence called the marker. These gene sequences are usually those which confer resistances to a range of antibiotics such a ampicillin, or nutrient (e. g., sugars such as arabinose) utilizing gene sequences.

In the present experiment, a gene for fluorescent green pigment characterized from a jelly fish was used to genetically transform Escherichia coli bacterium. The procedures used were standard but strictly as recommended by the commercial supplier of the gene. This gene was integrated into a plasmid called E. coli HB101 K12.   This was obtained from a commercial supplier (BIORAD) who also provided the all the other required material along with detailed step  by  step instructions for carrying out the bacterial transformation with the gene for fluorescence.

The supplied plasmid, pGLO contained the GLO gene extracted and purified from a mutagen treated jelly fish called Aequoria victoria. The property of the GLO gene is to produce an enhanced bright Green Fluorescent Protein (GFP) which produced a bright green fluorescence when exposed to ultraviolet light in darkness. This plasmid also contained the ampicillin resistance (AMP) gene. The host bacterium containing this plasmid (pGLO) has the ability to grow on a nutrient medium to which a sugar arabinose has been supplemented. This medium also containing the antibiotic ampicillin whose presence does not prevent bacteria from growing normally. This antibiotic resistance gene helps and serves as a marker for easy direct method for single step selective preferential growth of the bacterium containing the GFP along with AMP. The suggested and the expected results were formation and growth white colonies on the nutrient medium with the added ampicillin and arabinose. The real transformed colonies have the ability to grow and display the characteristic bright green fluorescence on arabinose medium and under UV light illumination in dark. This experiment of bacterial transformation is an example of real life experience.
But in the present experiment, none of these results were observed. The possible problems and the probable reasons for the lack of growth of bacteria obtained from our transformation experiment are discussed.  

Materials and Methods

    All the materials used were supplied by BIORAD Labs. The glassware, equipments and other disposables were used as exactly recommended and suggested by the supplier in the accompanying Instruction Bulletin of the BIOTECHNOLOGY EXPLORER TM  pGLO Bacterial Transformation Kit Catalog No. 166-0003EDU (

Materials and Methods

Kit Components Class Kit - 1. E. coli HB101 K 12, lyophilized in 1vial, 2. Plasmid (pGLO), lyophilized, 20 g in 1 vial, 3. Ampicillin, lyophilized, 30 mg in 1 vial, 4. L () Arabinose, lyophilized, 600 mg in1 vial, 5. Transformation solution (50 mM CaCl2, pH 6 sterile, 15 ml in 1 bottle, 6. LB nutrient broth, sterile, 10 ml 1 bottle, 7. LB nutrient agar powder, sterile (to make 500 ml) 1 pouch, 8. Pipets, sterile, individually wrapped 50 numbers, 9. Inoculation loops, sterile, 10 l, packs of 10 loops 8 pks, 10. Petri dishes, 60 mm in 2 sterile bags of 20 each, 11. 60 Microtubes, 2.0 ml, (10 each yellow, green, blue, orange, lavender, pink), 12. 8 Foam micro test tube holders, 13. Instruction manual 1

    The transformation experiment required additional equipments.These were as follows and were present in our laboratory.

    1. One UV lamplong wavelength (catalog  166-0500EDU) 2. One Clock to time 50 seconds 3.  Microwave oven (1), 4. Incubator oven set to 37 0 C, 5. Temperature controlled water bath, 16 liter (1), 6. Thermometer that reads 42 0 C 1, 7. one 1 L flask , 8. 500 ml graduated cylinder (1), 9. Distilled water (from supermarket), 500 ml , 10. Crushed ice and containers (foam cups) (18)11. 10 ml of bleach (household variety) (10 ml), 12. Permanent marker pens (48)
Methods (Procedure)

a. Preparation of Nutrient Agar - All the LB nutrient agar from the kit was added to 500 ml of distilled water in a 1 L or larger Erlenmeyer flask. The flask was swirled thoroughly to allow full dispersal of the medium in water and placed in microwave oven for boiling stage with swirling the flask repeatedly 3 times in between. The flask was cooled to about 500 C on bench top for some time and then poured slowly into previously labeled Petri dishes. Agar plates were prepared 3 days prior to starting the experiment and were left for 2 days at room temperature before storing them in refrigerator until use.

b. Preparing arabinose and ampicillin solutions  With a sterile pipetThree ml of sterile transformation solution (supplied) was added to arabinose containing vial and vortexed for thorough dissolution. Similarly 3 ml of the sterile transformation solution was added to vial containing ampicillin and mixed well. The vials placed such that the nutrient agar was not solidified. 

LB nutrient agar prepared earlier was poured into Petri plates (about less than half) labeled as LB 16 plates, 16 as LBamp, and 8 plates LBampara. Then dissolved ampicillin was added to the medium and shaken this was poured into other 16 plates labeled LBamp. Similarly, to the rest of the LB medium arabinose was added and medium was poured onto 8 plates labeled as LB amp ara. These plates were stacked inverted in 4-8 numbers and stored in a refrigerator till use.

c. Preparing bacterial cultures and pGLO plasmid.  E. coli bacteria (supplied as lyophilized) were rehydrated and allowed to stand at room temperature for 5 minutes.  The petri plates were streaked in quadrants of Petri plates by wire loops with this inoculum to obtain single colonies. Similarly pGLO plasmid solution was prepared using the 250 ul transformation solution (supplied).   

Transformation  One ml of transformation solution (CaCl2) and 1 ml of LB nutrient solution were taken in the supplied color coded and labeled microtubes.

    CaCl2 (250 ul ) was taken in two tubes each labeled as pGLO  and pGLO-. A sterile wire loop was inserted into pGLO plasmid (fluorescing green in UV light). A loopful of this solution was taken and put into transformation solution (labeled pGLO). This was kept on ice for 10 min. No pGLO was added to pGLO- tube kept on ice.

With a new sterile loop  bacteria from a single colony was collected and into inserted into the bottom of of the pGLO  tube. Similarly another sterile loop was used to insert bacteria into pGLO- tube. The loops were spun thoroughly in the tubes so that the bacterial mass was fully dispersed without showing any clumps.

These tubes were placed back on ice.

     With separate sterile pipets, 100 ul of each of these pGLO and pGLO- solutions were taken and uniformly skated on the surface of the appropriately labeled (LBamp plate pGLO LBampara plate pGLO  other LBamp plate -pGLO the LB plate-pGLO) agar plates for even distribution of the solution on agar surfaces. The Petri plates were stacked and incubated in an oven at 37 0 C.     

Heat Shock  using the foam rack as a holder, pGLO and pGLO- tubes were placed into a  water bath set to 42 0 C for precisely 50 seconds. After exactly 50 seconds, the tubes were shifted to ice very rapidly. The tubes were placed on ice for 2 minutes.

    The foam racks containing the two tubes were brought to bench top at room temperature. Using a separate fresh sterile micropipettes 250 ul of LB nutrient broth was added to each tube. Each was flicked thoroughly to allow uniform distribution. These tube were incubated at room temperature for 10 minutes. Using fresh and separate micropipets 100 ul of each solution was dispensed into appropriately labeled plates (pGLO  LB amp pGLO- LB amp ara -pGLO LB amp -pGLO LB. Using separately fresh and sterile loops the solution was evenly skated on the agar surfaces. The plates were stacked inverted and incubated in an oven at 37 0 C for overnight. The petri plates were observed the next morning.

The observations were on the growth of the colonies. Calculations were made on efficiency of transformation by using the counts of colonies formed, initial amount of DNA used. The colonies formed were also checked for fluorescence properties in the hand held UV lamp of long wavelength

All the necessary precautions were implemented in all the steps and stages in transformation. The transformed bacteria were incubated at 37 0 C for overnight. The only deviation from the recommended procedure was that instead of keeping the cultures in a water bath, they were placed in a heating block.   

    Surprisingly, there was no symptom of any growth of the transformed E. coli on the LB nutrient medium supplemented with the necessary concentration of arabinose as well as ampicillin. Therefore no data were available for carrying out the transformation efficiency and other paramerters.

    It was disappointing to observe the unexpected results of no growth in the colonies of the E. coli transformed with the pGLO plasmid also containing beta lactamase gene for conferring ampicillin resistance. The experiment has to be repeated but with extra precautions to avoid any mistakes done earlier.
    Many reasons and causes can be conceived. The procedure used in the present experiment deviated from the recommended one in the Instruction Bulletin in that the cultured bacteria were not placed in the water bath but placed in heat block. This may have caused too much of a sudden heat shock. It is well established that bacteria die beyond 420 C.  

    The other reason could be that the bacterial cells were not at the exact right stage (just before the log phase) for their readiness to be used to make them competent. Chemical methods of transformation are known to be not so efficient. Dury (2008) recommended the use of electroporation because chemical methods as used in the present experiment are not high enough to produce good desirable levels of transformation efficiencies.  Similar suggestions were made by many other researchers on many other systems (e. g., Alegre et al 2004).

    Importance of plasmid size and the DNA size may another important parameter for low efficiency of or no transformation. Szostkova and Horakova (1998) observed that the frequency of transformation is inversely related to the DNA and plasmid sizes.

    Many other reasons may be assigned as follows
The plasmid may have been of poor quality
The plasmid may have been unstable under the conditions received and stored
A good and effecting of the heat shock may need to be checked again
Sterilizations methods used may have been very harsh
Incorrect or very antibiotics used
Preparations of the stocks and their final solution mixtures may not have been    correct.

    As no colony formation was observed the experiment needs to be repeated with extra precautions. Also the results as observed will also be communicated to the supplier for further advice and guidance on repeating the experiment.

    In the repeat experiments more experiments have to be performed by using different transformation protocols including chemical and physical (electric) methods. In the chemical methods, it may be necessary to use different modifications of temperature levels, durations, the number of shocks. It may also be important and useful to use bacteria at different times during log and lag phases. This would help in catching the bacteria at the very time of their sensitivity and amenability to transformation methods.


I. A missionary couple, living in West Africa brought their 4-year old son to the office of their physician on the second day of a visit home to Minnesota. The boy had severe  continuous diarrhea about seven hours earlier. His mother described his diarrhea as whitish with flakes in. There was blood however when a blood film was made whole blood cell could be identified.
1. This disease is most likely caused by what bacteria and what is the name of the disease
The disease is most likely caused by the bacterium Vibrio cholerae and the disease is Cholera.
What would confirm your choice
To confirm my choice, I would perform a laboratory tests that include the stool gram stain (gram negative rods) culture dark field microscopy or stool Polymerase Chain Reaction (PCR), depending on the testing kit available.
The boy had all of his vaccines so why does he have such an infection
This is because Cholera has no vaccine, thus one is susceptible to it.
What course of treatment would you order
I could order rapid intravenous replacement of the lost fluid and ions. Then followed by administration of isotonic maintenance solution (Oral Rehydration Therapy) continuous until the diarrhea ceases.
What could be the likely source of this infection
Likely source of infection could be through consumption of raw or undercooked shellfish, water or food, contaminated with V. cholerae.
II. A 7 weeks baby girl was brought into the hospital with a rash on hands and feet, fever, cough and high WBC count. The baby was premature 35 weeks at a weight of 2.97kg. She was treated with Penicillin G for 14 days  testing after that showed the treatment was successful. The parents were treated too.
What infection did she have
      She had congenital syphilis.

Name the causative agent.

Causative agent is the bacterium Treponema pallidum.

How did she contract this microbe

She contracted it through transmission from the mother during pregnancy as blood containing the bacteria reached the fetus through the placenta, the organ that nourishes the developing baby.

At what stage of the infection was she, and why

She was at the secondary stage of the infection. This is because most infants born with syphilis have no symptoms of the disease. Almost all develop symptoms by three months of age, which according to the symptoms she had it is already in the secondary stage of syphilis. The primary stage symptoms include a chancre where the bacterium entered and enlarged lymph nodes at the groin, indicating that the syphilis is in the secondary stage. 

Why were the parents treated too

The parents were treated because they had contracted the disease that was eventually transmitted to their daughter. Hence, the diagnosis was to prevent further infections in the family.

III. A 24-year old female presents herself at the doctors office. She complains of fever and of pain in her digestive tract. She had diarrhea that was bloody. A blood film was made of her stool and no blood cells were noticed. Physical examination showed the patient had a tender abdomen. She had eaten at a fast food place 4 days ago  she felt ill for the past couple of days, she had vomiting and fever too. Microbial analysis showed gram negative non-spore forming rods. 
What type of food disease do you believe she has
She has Shigellosis a form of food infection that may result when foods containing Shigella bacteria are consumed.
2.  What are the factors that assist you in your identification of the causative agent and why
The factors that assisted me in identification of the causative agent are the physical examination that revealed a tender abdomen bloody diarrhea that had no blood cells analysis that showed gram-negative non-spore forming rods and the incubation days for the symptoms to appear. These were vital in differentiating the causative agent from Vibrio cholerae, E. coli, Salmonella, Yersinia entercolitica and Campylobacter fetus, which are related to Shigella.
3. What agents (at least 3) you would not consider  why
I would not consider Vibrio cholerae, because the microbial analysis did not show diarrhea as whitish with flakes nor blood film showing presence of whole blood cells. I would also rule out Salmonella and E. coli, because their symptoms do not include bloody diarrhea.
4. What treatment will you advise  why
I would advise a bactericidal antibiotic for instance metronidazole Oral Rehydration Therapy (ORT) for treatment of dehydration and antispasmodics if at all the cramps are severe. This is because virulent Shigella strains produce disease after invading the intestinal mucosa the organism only rarely penetrates beyond the mucosa, thus the bactericidal will kill the virulent Shigella strain that produces the disease. The ORT is to restore the lost fluid and antispasmodics are to relieve the pain.
5. What is the virulence factor in such an infection
Shigellae do not produce enterotoxins and they are not haemolytic. As a prerequisite of Shigella virulence, the bacteria have to penetrate into the intestinal epithelial cells and proliferate there. This is penetration is arbitrated by a Shigella-induced phagocytic event. The products of the genes encoded in the large virulence plasmid as well as some chromosomal genes are involved in this step.
IV.   A 27-year old white female presented her self at her local physician on August 15. On physical exam, the patient had a fever of 38.5C. She appeared fatigued, had tender abdomen and complained of headache, stiff neck and backache with diarrhea. The physician noted an irregular heartbeat. The patient complained of lack of ability to concentrate. The patient gave the following history She is a graduate student in the wildlife program at the University in her town. She was in the field for three weeks in Wisconsin during the months of May and June. She tracks small mammals in the field and studies their behavior. It had been a warm, wet spring and she complained of a large number of flies in the area. She felt sick the last day there after eating some canned food, as there was no cooler there. You had no time for culturing, as you need to take action fast.
What is your best diagnosis of this case
My best diagnosis of this case could be treatment with beta-lactam antibiotics such as ampicillin andor the addition of gentamicin frequently for its synergistic effects. Also I could administer intravenous or oral rehydration therapy to control dehydration, and some pain killers to relieve fever and pain.
What features are critical to your diagnosis
The features that are critical to my diagnosis are Headache, stiff neck, diarrhea, fever and irregular heartbeat.
3.   What further steps should be taken to precisely identify the case  why
Further steps to be taken is to culture for pathogens from the blood or Cerebrospinal fluid, this is because of lack of reliable serological or stool tests to test for my assumed pathogen . The whole issue is to establish the causative agent which I suspect to be Listeria monocytogenes, because the central nervous system (CNS) has been affected resulting into a stiff neck and loss of concentration.
4.  What microbes you will not consider and why name 2.
I will not consider Clostridium botulinum and Bacillus cereus. This is because their symptoms dont include a stiff neck which is symptomatic for meningitis, as a result of CNS infection with Listeria monocytogenes.

1.  What are parasites and their virulence factors List 4 diseases that are parasitic.
Parasites are organisms that derive all of their nutrients from other organisms called hosts. The parasites can be either ecto-parasite or endo-parasite. The parasites dont contribute anything to the host and thus they harm the host or can even kill it in some cases. Parasites possess surface antigens with amino acid repeats. Making up the interface between host and pathogen such repetitive proteins may be virulence factors involved in immune evasion or cytoadherence. Diseases that are parasitic are Malaria caused by the endoparasite protozoan Plasmodium spp. Trichomoniasis caused by the endoparisite protozoan Trichomonas vaginalis Toxoplasmosis (Parasitic pneumonia) caused by the endoparisite protozoan Toxoplasma gondii and Schistosomiasis (bilharzia) caused by Schistosoma sp.
2.  Do you think that recombinant protein vaccines could work for HIV, why
No Recombinant protein vaccines can not work for HIV. This is because, HIV is highly mutable, this is,  the virus has an ability to swiftly counter to selective pressures imposed by the immune system, the count of virus in an infected individual typically evolves so that it can evade the two major weapons of the adaptive immune system humoral  and cellular immunity. Also, HIV isolates are themselves highly variable, this is, it can be categorized into multiple clades and subtypes with a high degree of genetic divergence. Therefore, the immune responses raised by any vaccine need to be broad enough to account for this variability. Any vaccine that lacks this breadth is unlikely to be effective.

3.  Name 2 bacterial  2 viral STDs and their causative agents. Name 3 possible symptoms that may indicate an STD infection
Two bacterial STDs are Syphilis and Gonorrhea. Two viral STDs are Genital Warts (HPV) and Herpes. Three symptoms that may indicate an STD infection include Burning or pain during urination, unusual vaginal or penile discharge, and blisters or sores on the genitals, anus, or surrounding areas.
4.  Name the most identifying feature for each stage of syphilis. What do you understand by the term Latency period
The primary stage (first stage) is identified by appearance of a chancre. The secondary stage disseminated stage (second stage) is identified by appearance of allergic like hives at the palms of the hands and soles of the feet. The tertiary stage or late stage is identified by infection of any body tissue. The term Latency period is the period between secondary and tertiary stages of syphilis, it can last for many years before the tertiary or late stage evolves.

5. What is diarrhea  why diarrhea is important in identifying food infections  Give 3 examples of food illness that could be identified via their diarrhea

Diarrhea is loose stools that last for several days or even more. Diarrhea is important in identifying food infections, because the characteristic of the stool, for instance color and its contents like mucous and blood depicts a specific type of food infection. Three examples of food illness that could be identified via their diarrhea are Cholera, Shigellosis and Salmonellosis.

6.  What is Epidemiology and how could it be used to prevent the spread of disease

Epidemiology is the study of the distribution and determinants of health-related states or events in specified populations, and the application of this study to the control of health problems. Epidemiology could be used to prevent spread of disease through searching for causes and other factors that influence the occurrence of health-related events such as diseases, syndromes, and injuries. This can then be used in comparing groups with different rates of disease occurrence and with differences in demographic characteristics, genetic or immunologic make-up, behaviors, environmental exposures, and other potential risk factors. Under ultimate circumstances, epidemiologic results provide sufficient proof to direct swift and effective public health control and prevention measures.

7.  How does the toxin action of Clostridium botulinum differ from that of Clostridium tetani

The toxin of Clostridium botulinum is absorbed by the upper part of the gastro-intestinal tract in the duodenum and jejunum, and passes into the blood stream by which it reaches the peripheral neuromuscular synapses. The toxin binds to the presynaptic stimulatory terminals and blocks the release of the neurotransmitter acetylcholine, which is required for a nerve to simulate the muscle. Active Clostridium tetani cells at the tissue level releases an exotoxin called tetanospasmin that causes certain nervous system irregularities by means of retrograde transmission through neurons to the brain. One of the toxins effects includes constant skeletal muscle contraction due to a blockage of inhibitory interneurons that regulate muscle contraction. Tetanospasmin initially binds to peripheral nerve terminals, then are transported within the axon and across synaptic junctions until it reaches the central nervous system. There its rapidly fixed to ganglion sides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis. The toxin blocks the release of inhibitory neurotransmitters across the synaptic cleft these are required to check the nervous impulse. If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus. Tetanospasmin appears to act by selective cleavage of a protein component of synaptic vesicles this prevents the release of neurotransmitters by the cells. Binding appears to be an irreversible event.

8. What are the main microbial difference between Listeria monocytogenes  Brucella melitensis  their infections

Listeria monocytogenes is non-spore gram-positive rod-shaped bacterium. It is ingested with raw, contaminated food. An invasion toxin secreted by the pathogenic bacteria enables the pathogen to penetrate host cells of the epithelial lining, the infected person experiences vomiting, nausea and diarrhea. It causes Septicemia and meningitis in patients with suppressed immune infection. It also causes listeriosis, which is an inflammation of the brain. While Brucella melitensis is an aerobic coccobacillus gram-negative, non-motile, and non-spore forming bacterium, which enters the body via the skin, respiratory tract, or digestive tract. Once there, this intracellular organism can enter the blood and the lymphatic where it multiplies inside phagocytes and eventually cause bacteremia. Symptoms include fever, headaches, chills, depression, profound weakness, arthralgia, and weight loss.

Brucella melitensis is odd in numerous ways. First, the bacterium does not bear classic virulence factors, such as capsules, secreted proteases, exotoxins, endotoxins, pili andor fimbriae or virulence plasmids, and its lipopolysaccharide pathogenicity is not typical. Second, it exhibits a tendency to invade and persist in the human host through inhibition of involuntary cell death. Its virulence depends on survival and replication properties in different cell types in which Brucella melitensis controls the maturation of its vacuole to avoid innate immune responses and to reach its replicative niche related with the endoplasmic reticulum. While for Listeria monocytogenes, the invasion toxin secreted by the pathogenic bacteria enables the pathogen to penetrate host cells of the epithelial lining. It multiplies not only extra-cellular but also intracellular, within macrophages after phagocytosis, or within parenchyma cells which are entered by induced phagocytosis. After engulfment, the bacterium may escape from the phagosome before phagolysosome fusion occurs mediated by a toxin, which also acts as a hemolysin, listeriolysin O (LLO). The bacterium stimulates a CMI response that includes the production of TNF, gamma interferon, macrophage activating factors anda cytotoxic T cell response. Possibly, in humans, a failure to control L. monocytogenes by means of CMI allows the bacteria to spread systemically. Additionally, unlike other bacterial pathogens, Listeria is able to penetrate the endothelial layer of the placenta and thereby infect the fetus.


As the name suggests, DNA sequencing is simply the process of determining the content and arrangement of nucleotides in a particular genome, either in part or in whole. Most DNA sequencing techniques are based on the dideoxy method which was first described by Sanger et al (cited in Pierce, p.553-5 Davies, 2001, p.10). The dideoxy method of DNA sequencing is based on the principle of replication. After the DNA of interest has been amplified using the polymerase chain reaction (PCR) method, the amplified products are divided into 4 different tubes and subjected to denaturation. The resultant single strands or the primers complementary to the single strands are labelled and the DNA subjected to further rounds of replication (Pierce, p.554).
However, these subsequent replication rounds generate many short contigs since dideoxyribonucleotides (ddNTPs) are used in place of deoxyribonucleotides. The ddNTPs do not have a free 3 OH group and therefore termination of replication occurs whenever any of the 4 ddNTPs is incorporated into the growing chain. Each of the 4 different ddNTPs is added into one particular tube. Separation of the DNA fragments through gel electrophoresis followed by autoradiography reveals sequentially arranged bands which can be easily read to determine the sequence of the DNA of interest (Pierce, p.554).
In recent times, the dideoxy method has become highly automated and high tech with variants such as massively parallel signature sequencing (MPSS), expressed cDNA sequence tag (EST), and serial analysis of gene expression (SAGE) techniques vastly improving the turnaround time, quality and throughput of DNA sequences (Mount, p.33).
As in health, DNA sequencing has found many applications in the food industry. This essay looks at the impact of rapid sequence DNA on food with a particular emphasis on how the data generated is handled, how it can inform our understanding of underlying mechanisms, what the future holds among others.
Impacts of Rapid DNA Sequencing on Food
DNA sequencing has had a significant impact on food. First, it has led to an improvement in the safety of food. This is primarily because the technique allows the detection of food borne pathogens with stunning accuracy. Common pathogens which have been implicated in food borne illnesses include the 0157H7 strain of E. coli, Campylobacter species, Listeria species and Salmonella (Volokhov, p. 4270-8).
The challenge to food safety is particularly serious when it is considered that these pathogens are not only pervasive in the environment but are also spread through innumerable reservoirs. While the pathogenicity and pervasiveness of these pathogens is widely documented, DNA sequencing techniques have in particular helped to improve the sub-typing of species and this has made it possible for pathogenic organisms to be identified at the subspecies level (PhysOrg).
Another impact which rapid DNA sequencing has had on food relates to quality control. The technique has been widely employed to assess genetically modified (GM) contamination in food. In this instance, it is a routine practice that is commonly applied in many countries, including the UK, to assess whether there is any GM contamination in food in order to protect the public from potentially undesired effects. This is because GM food has been implicated in horizontal gene transfer and has been noted in some instances to cross the species barrier. Foods which have been analyzed through rapid DNA sequencing to determine their purity include potatoes, rice, tea, coffee and Drum wheat. However, PCR is reportedly ineffective in discriminating GM from non-GM content (USDA PhysOrg).
Additionally, DNA sequencing has been used to improve the nutritional value of foods. Examples of foods which have been enhanced through this technique include rice, soya, and maize. Specifically, these foods have been improved through recombinant DNA technology which utilizes DNA sequencing in primer design and to confirm that transformation has indeed taken place (Barnaum, p. 69-118).
Rapid DNA sequencing techniques have also helped to improve food security. Globally, food security is under threat due to the effects of climate change, desertification, a growing human population, poor agricultural practices among others. For example, there has been a significant reduction in the yield of harvested wheat globally due to climate change (Physorg). Rapid DNA sequencing techniques can help to boost the food security especially as regards important food crops. According to Physorg, scientists in the UK have embarked on a major project to determine the genetic sequences of several varieties of wheat. This project is expected to enhance and provide new knowledge on how the genes for wheat are expressed, discover disparities in gene networks that are of vital importance to resistance to diseases and improve the yield and hardiness by discovering genes responsible for these traits (PhysOrg).
Yet another impact of rapid DNA sequencing on food is that it has led to significant cost saving. This is because it has reduced the incidence of food borne pathogens which have been associated with huge economic losses in many countries throughout the world. Enhanced human health is another area that has been impacted upon by rapid DNA sequencing methods. Due to rapid DNA sequencing, the turnaround time required for the diagnosis of diseases caused by food borne pathogens has been reduced. Additionally, newer and more effective targets have been identified, leading to more effective diagnostic and therapeutic strategies aimed against the food borne pathogens and effective. Important also is the fact that people are eating food that is less hazardous and safer as a result of DNA sequencing techniques (Barnaum, 69-118).
Rapid DNA sequencing has also improved our understanding of diseases caused by food-borne pathogens. Biodiversity, especially as it pertains to plant and animal food species, is another area which has been impacted by rapid DNA sequencing. Due to this technique, preservation of whole DNA sequences of plant and animal food sources is possible. Besides, rapid DNA sequencing methods have made it possible to preserve the genomes of food species that are in danger of extinction. This will enable the species data to be made use of long after they have become extinct (Barnaum, 69-118). 
How the Sequence data is handled
Sequence data obtained through rapid DNA sequencing techniques is stored in databases. Currently, many databases exist to store the sequenced data and any other information pertinent to the sequences. Bibliographic databases such as AGRICOLA, BIOSIS, and PubMed contain important bibliographic data related to food. Sequence data is also stored in taxonomic and nucleotide sequence databases. Examples of taxonomic databases include. NCBIs Taxonomic Browser and the Tree of Life. Nucleotide sequence databases include the DNA Data Bank of Japan (DDBJ), the European Bioinformatics Biotechnology Laboratory (EMBL) which is hosted by EBI at Hinxton in the UK, and GeneBank. Secondary databases which are non-redundant and curated include Refseq, CCDS, dbEST which is a database of expressed sequence tags, dbSNP which is a database of single nucleotide polymorphisms, UniGene among other. Third party annotation (TPA) databases, genomic databases, protein databases, structural databases, gene expression libraries and micro array databases are also used to archive data obtained through rapid DNA sequencing (NCBI, 2009 Rashidi  Buehler, 2000). .
Examples of genomic databases include the Proteome Analysis Database and the Online Mendelian Inheritance in Animals (OMIA). UniProtKBSwiss-Prot is an example of a protein database that has entries from many plants, microbial and animal species that have an impact on food security, availability and safety or which are sources of food such as Arabidopsis thaliana, salmonella typhimurium, and E. coli. Secondary structural databases such as PROSITE, pfam and PRINTS are also of immense value. Other types of databases which contain sequence information that is pertinent to food are structural databases such as the Protein Data Bank (PDB), micro array databases and gene expression libraries (GEO) (NCBI, 2009).
The databases have dedicated tools that can be used to submit the sequenced data. Examples of such tools include the SAKURA, Sequin or the Mass Submission System (MSS) in DDBJ, SPIN in UniProt, and AEdb ENA or IMGTHLA links in EBI. Microaarray submissions are sent through the ArrayExpress link. High throughput genomic sequences are sent through an FTP link for all the major databases including GenBank, EMBL and DDBJ (NCBI, 2009).
Use of the 3 primary databases is free and is coordinated by the International Nucleotide Sequence Database (INSD) collaboration. According to the rules governing these sequence databases, anybody can submit sequence data at anytime for free, the data is available to any interested party at no cost, no restrictions to access the databases are placed, sequences can be corrected by the submitters but all sequences which have been submitted remain accessible through their accession numbers, and full disclosure is made to the public on the submitted sequence data (NCBI, 2009).
How the knowledge can help to advance the underlying mechanisms
The knowledge stored in these databases can be used in many ways to advance the knowledge of underlying mechanisms. First, the information can be used for gene expression studies. One particular important use of DNA sequence data is in determining the manner in which genes are expressed. Gene expression can be done through current sequencing methods such as MPSS, EST sequencing and microarrays (USDA, 2009 Stekel, 2003).
The information can also be used to predict gene function either through ab initio methods or through homology based search techniques. In homology based searching, an example would be determining the function of a gene through comparison of this gene with other similar genes whose function has been elucidated before. Similar methods can also be used to determine the protein function and metabolic pathways of DNA sequences (Eisenberg, Marcotte, Xenarios  Yeates, 2000 Blanco  Guigo, 2005).
Additionally, the information can be used to determine the evolutionary mechanisms through phylogenetics. Here, the evolutionary changes in DNA sequences of related food-borne pathogens, plants or animals used for food are compared. This will enable the generation of more accurate evolution mechanisms as it does not depend solely on physiological features or physical taxonomy. Whole genome sequences can also be compared and intricate evolutionary occurrences, for instance horizontal gene transfer, studied. The obtained sequence data can also be used to come up with computation-based models which would help to predict the long term prospects of the system under consideration. This can be accomplished in 4 main steps and these include multiple sequence alignment, determination of the substitution model to use, creation of phylogenetic trees and evaluation of the tree using bootstrap analysis, likelihood ratio tests, permutation or skewness tests (NCBI, 2009).
Of particular importance has been the use of this DNA sequence information in the design of primers. The sequence data has been used to create probes for hybridization and this has proved useful in identification of food borne pathogens. Sequence analysis can be used to detect mutations in microbes affecting food and in food sources such as plants and animals. Currently, there is a lot of sequence data and methods such as the comparative genomic hybridization and single nucleotide polymorphism arrays have been developed to identify and locate point mutations (NCBI, 2009). This is especially important for food-borne pathogens and animal and plant foods and will help to identify mutations responsible for drug resistance in the pathogens or lethal mutations that can cause reduced yields and death of plant and animal species. However, the data is generally noisy and is thus not very useful. The information obtained from rapid DNA sequencing can also be used in the high throughput analysis of proteins, genome annotation and to analyse the way regulation is carried out by plants and organisms which serve as food sources or which affect the availability or quality of food in one way or another (Kolchanovv  Hofestaedt, 2004).
Currently, micro arrays are being utilized to simplify the analysis of contaminated food. Here, thousands and thousands of sequences for different pathogens are immobilized on a platform such as a silicon chip. This enables the simultaneous identification of pathogens that can potentially cause a food borne disease, the drugs which it is resistant to and its pathogenicity. This has therefore made it simpler to analyze contaminated food (Stokel, 2003).
The success of DNA sequencing methods on food has largely been as a result of the availability of highly precise and discriminative oligonucleotides and the complete automation of the DNA sequencing technique. Regarding automation, machines which utilize robotics to provide automated pipetting and highly mechanized gel loading systems, bar-coded comb loading and cycle sequencing have helped to significantly reduce the turnover time besides enhancing the quality and quantity of the outcomes obtained (ScientistLive, 2009).
Multi locus sequence typing (MLST) has also been applied in the DNA sequencing of food with relative success. The MLST technique is different form techniques which are based on banding patterns in that the nucleotide sequences variations in multiple allelic genes which manifest low mutation frequencies are targeted. The technique can be replicated easily and has been used to distinguish outbreaks of Listeria (Zhang, Jayaro  Knabel, 2004). However, the technique is unable to discriminate between different isolates of the 0157H7 strain of E. coli since the homology of the allelic genes is nearly 100 (Chang, Zhang  Knabel, 2005).
Future of DNA Sequencing in Food
Whereas the widespread application of DNA sequencing has vastly improved the quality and availability of food, there are some inherent challenges associated with the current DNA sequencing methods. A particularly important hindrance is that the information which is obtained through DNA sequencing, especially if it is high throughput data, is associated with a lot of noise. This noise has reduced the utility off some of the available DNA sequencing techniques. Future use of DNA sequencing is thus premised on newer tools which are geared at reducing this noise (Bansal, p.1-11).
For instance, studies of gene expression using currently available techniques are generally deemed to be full of noise and are largely subjective. The focus of future studies is on the development of statistical techniques which can separate the noise for the gene sequences from useful signals especially in high through put gene expression studies (Bansal, p.1-11).
Tools based on the Hidden Markov model and change point analysis are also being developed so that the large quantity of sequence data currently available can be effectively utilized to study mutations in plant and animal food species and food-borne pathogens  (Bansal, p.1-11).
Additionally, studies of protein-protein docking also promise better prospects in future. It is expected that protein-protein interactions of the 3D structures can be elucidated based solely on these structures thus obviating the need for wet laboratory. If this happens, it will be possible to develop highly effective and low cost diagnostic and therapeutic tools directed against food-borne pathogens quickly and with less difficulty  (Bansal, p.1-11 Blundell, p.413-23).
In future also, high throughput image analysis may become a possibility and can be used in many ways to boost food security. A commonly cited example which is applicable to food is the inference of contigs in DNA mapping.  Rational drug design through lead optimization that is based on analysis of DNA sequences is yet another possibility. Other future trends include whole genomic expression analysis using microarray hybridization, increased commercialization and therefore use of gene chip technology and improvement of multi-locus sequence typing methods (Blundell, p.413-23 Bansal, p.1-11).

The DNA sequencing technique has been used widely in the food industry. The technique has had a great impact on food. It has helped to enhance food safety, improved the nutritional value of food and enhanced food security. Use of rapid DNA sequencing has also aided in the quality control of food, led to cost savings, improved the understanding of food borne diseases and led to the development of better diagnostic techniques against food borne diseases.
There is a lot of sequence information and this is stored in bibliographic, nucleotide, genomic, protein, and microarray databases. The information is used in gene expression studies, prediction of gene function, prediction of protein function, primer design and elucidation of evolutionary mechanisms. Success of the technique has primarily been due to automation and development of highly specific and accurate oligonucleotides. Future prospects are focused on reducing the amount of noise inherent in the data through hidden Markov Model (HMM) tools, protein-protein docking, high throughput protein analysis, lead optimization, whole genomic expression analysis using microarray hybridization and increased use of gene chip technology.


Antibiotics are the drugs which are prescribed by the physician to fight against infection caused by microorganisms.Antibiotics are documented as agentsto treat illnesses. But there are instances whenantibioticsarespecifiedby physicians to prevent infections.
Usually antibiotics are given prophylactically in certain medical conditions only, like when a person has transplanted heart valves or damaged heart valves. These people are more prone to develop cardiac heart valves infections even with minor surgeries . This is due to the lodging of bacteria from other sites of the body through the blood stream into the abnormaldiseased heart valves during the surgical process. The physicians therefore prescribe antibiotics propylactically to people with heart valves problems who are even undergoing dental surgeries.Antibiotics are also prescribed to indivisuals who are immunocompromised especailly people who have AIDS and are also preferred in patients undergoing chemotherapy and radiation therapy.Persons who are immunocompetent are also sometimes prescribed antibiotics before a major surgery like bowel surgery. People travelling to third world countries are given prophylactical antibiotics to prevent them from prevailing illnesses in the third world countries, like diarrhea.
It is recommended that it should only be the physician right to decide whther antibiotics is needed by the indivisual for the infection or not. In ordinary infections, it is not advisable to start antibiotics at all.It should be concised that the improper and excessive use of antibiotics lead to antimicrobial resistance.

Overuse and development of resistance
    Antibiotics are not recommended for a normal cold, because antibiotics only work against fighting infections caused by bacteria. Viruses cause illnesses such as a soar throat cold or flu. Antibiotics do not cure infections caused by viruses and should not be taken in these cases. It is advised that a cold or the flu be allowed to run its natural course. The average cold or flu lasts up to two weeks or more. If the symptoms persist for more than four weeks then it would be wise to consult the physician. In most instances, antibiotics either kill bacteria (bacteriocidal drugs) or cause their growth to come to a halt (bacteriostatic drugs). However, in some situations, certain bacterium grows stronger than the antibiotic coverage and the drug is unable to be effective aginst them. These strains of bacteria are called resistant, as they are in fact resistant to antibiotics.
Usually resistance to antibiotics develops when a person has taken antibiotics either too often or improper dosages. Another form of mistreat is by patients who insist antibiotics prescriptions from their physicians. Remarkably, many doctors concede to misguided patients who stipulate antibiotics to treat colds and other viral infections that cannot be cured by these antibiotics. Due to the malpractices,there are certain bacterias which are resistant to various antibiotics and are termed as multi-drug resistant. Though we cannot completely stop resistance from occurring, we can slow down its progress and hinder its spread.Patients and physiciansmust take additional care, caution and consideration while making use of the antibiotics.
Individuallyif there is no serious infection in the body and we dont require antibiotics, we should not take them. Many infections are cured without antibiotics and it is not at all compulsory to use antibiotic in minor ailments.Bacteria are becoming increasingly resistant and there arent too many new antibiotics in the pipeline. It is therefore strongly recommended to use antibiotics optimally in order to seize the emerging bacterial resistance. The people need to be informed that by not using the antibiotic unnecessarily, the drugs will more likely to work when it is needed at the time of serious bacterial infections.
The future of antibiotics
The future of antibiotic is desolate. There is a marked decrease in the indurty of Research and development and without extensive researches and preventive practices the risk of resistance against antimicrobial drugs can not be addressed. The processes by which bacteria exhibit resistance to antimicrobials are usually drug inactivation or modification, alteration of target site, alteration of metabolic pathway and reduced drug accumulation.
Antibiotic resistance is trule a multi faceted problem and a challenge for public health. . The extensive utilization of antibiotics in medicine has a significant role in the emergence of resistant bacterial species.For controlling the developing resitance many aspects of the society needs to be adressed  such as prescribing behaviour, guidance to doctors and health economy to name a few.
Extensive efforts are required to develop new antibiotics and patterns of prescription are to be changed otherwise the future of antibiobiotic arena is bleak

Antibiotic Efflux pumps in Prokaryotic Cells Assignment

1.  The types of membrane transporters are systematically organized in the prokaryotes and also found in eukaryotes with different characteristics, they include 2.A.1 Major Facilitator Super-family, 2.A.6 Resistance Nodulation Division, 2.A.7 Drug metabolite transporters, 2.A.17 Proton-dependent Oligopeptide transporter, 2.A.60 Organo Anion Transporter, 2.A.66 Multidrug Oligosaccharidyl-lipid Polysaccharide Flippase, 2.A.74 Multidrug Endosomal Transporter, 3.A.1 ATP Binding Cassette.
2.  Some of the major physiological mechanisms that the bacteria have developed to elude harmful molecules e.g. antibiotics and chemotherapeutic agents are the efflux pumps. These mechanisms were developed by the bacterial cells to expel toxic molecules found inside the cytosol and inside the cell. Some of these molecules might also include biological molecules. Other mechanisms along this are target modification and production of enzymes to neutralize antibiotics.
3.  The natural function of the membrane transporters is to facilitate the movement of molecules macro and micro molecules. This mostly may include biological molecules such as proteins and also other diffusible molecules that might enter the cell membrane of the bacteria. This is important for the cell to be able to maintain osmotic balance in the cytosol.
4. The major antibiotic transporters found in bacteria are Major Facilitator Super-family, ATP Binding Cassette, Resistance Nodulation Division, Small Multidrug Resistance and Multi-Antimicrobial Extrusion. The drugs acted on are not necessarily structurally or functionally related to each other. Due to the ability of the super-families to recognize different drugs that are not structurally similar, leads to the strain ability to develop cross-resistance that may be the genesis of multi-resistant strains an example P.aeruginosa.
5.  NO.  Different organisms have the capabilities of expressing more than one type of transporter that may be similar to one expressed by another organism. These transporters are known to occur in more than one species. This is because the efflux pumps are known to be located in the bacteria plasmids or transposons and are then spread to other organisms through conjugation.
6. A drug can be recognized by different pumps. An example of the drugs is the tetracycline, fluoroquinolones and chloramphenicols. However those drugs within the same class tend to be more specific in the type of pumps by which they are recognized.
7.  Efflux pumps by themselves are only known to have the ability to give only low or moderate resistance. This is because the natural resistance of many bacterias is dependent on the active systems inducible expression. It is also known that for the efflux pumps to confer high resistance corporation between them and other resistance mechanisms is paramount. An example is resistance in P.aeruginosa whose high resistance is result from combination of, the active efflux system MexAB OprM, the outer membrane barrier and AmpC -lactamase. Further the antibiotics play an important role in the expression of the efflux pump during gene transcription and it s the one that induces the efflux pump.
8. Since genetic characterization has been successfully employed in identifying efflux resistance mechanisms in some bacteria, it has been suggested that genotypic tests be used to identify new effective antimicrobial drugs. Having knowledge of the mechanisms used by these organisms to promote resistance in them, may help in developing new drugs that are not easily susceptible to the efflux pumps of the organisms, thus lowering the probability of resistance to the drugs. This is because the manufactured drugs will be able to minimize over expression of the pumps in the organisms and the development of poor substrate derivatives.
9.  The development of new antibacterial drugs will be dependent on the knowledge of the actions of the pumps on the detection of the different drug molecules. Despite the discovery of pump inhibitors used for therapy, it is important that the new drugs are designed in such a way that they are broad-spectrum inhibitors acting on both gram-negative and gram-positive bacteria of different phylogenetic families to evade rapid developing resistance.


a) Zoonosis is a normal disease of animals that can be transferred to humans with or without the help of diverse vectors.  b) Vectors are living transporters of infectious agents. They can transfer infectious agents like bacteria, viruses and protozoans to other animals and humans. Popular vectors are parasitic arthropods and domestic animals. c) Arthropod-borne diseases are diseases caused by infectious agents that arthropods served as vectors. Arthropods are invertebrate animals which have segmented bodies, possess exoskeletons and have jointed limbs. Some examples are arachnids, insects and crustaceans. d) Reservior hosts are main hosts of an infectious agent but they do not manifest the clinical symptoms of the disease and they can transfer the infectious agent to other animal hosts.
The ecological factors involved in the efficacy of WNV transmission to humans include climate change alterations in temperature abundance of avian species reservoir and, increased numbers of vectors.  Temperature is among the factors that highly influence the transmission and propagation of viruses in their different hosts. The alterations of temperature to that of the optimal temperature for transmission and replication of the virus can increase the incidence and intensity of disease.  Vast numbers of avian species reservoir and insect vectors could also magnify disease outbreaks.
The crow or Corvus spp. is an ideal indicator of West Nile Virus (WNV) to humans because of four reasons. First, they are easily infected by this virus and these infections have a high mortality rate. Second, crows are everywhere and they can be easily identified. Third, they are major sources of WNV infections because the virus intensively multiplies in the crows blood making it easy for mosquitoes to contact the virus and then infect humans. And fourth, its convenient to collect samples from crows because it does not consume any resources and great amount of time.
In order to calculate the point and interval approximations of WNV disease risk in an indivuals  country residence based on DCD 0.1 (dead crows per square mile), the Cochran-Mantel-Haenszel (CMH) option of the freq procedure in SAS System for Windows V8 was employed.  Comparison of WNV incidence risk between the areas with DCD reports and those without DCD reports was made through the application of CMH chi-square statistic. Since it takes about 2-14 days before WNV infection will manifest in human hosts, detach computations of CMH-pooled risk estimates for 3 exposure periods were done. These 3 exposure periods are two weeks prior to, one week prior to and the week of observance of WNV human cases.
Real time monitoring can be advantageous to the prevention of disease outbreaks. The information generated from this system can signal the prompt employment of numerous measures to prevent disease outbreaks in humans. On the other hand, real time disease monitoring is a very complexed system and various variables can influence its effectiveness.  Any negative influence from the variables involved can be detrimental to the systems effectiveness.  An example is its dependence on voluntary reports only, if absence of participants to report the sightings of dead crows occurs in a certain area, this would translate to failure of WNV human infection risk prediction in that area.
The factors that play a role in limiting the effectiveness of the Dead Crow Density (DCD) index as a predictor of potential human transmission cases are the variations in disease vector among the areas involved and the area residents participation in the reporting of the dead crow. Disease vectors are important in the zoonoses, transfer of infectious disease from animals to humans. Significant reduction in the number of vectors involve can also significantly lower the disease incidence and intensity. One limitation of DCD index then is its failure to consider the role of vectors in the transmission of disease. To reduce the impact of vectors in the study, a calculation variable for vectors could be included in the statistical analysis. Another limiting factor is the participation of people in reporting the sightings of dead crows. Low reporting participants translate into low specificity of the calculations. Hence, a solution is to educate and encourage the people to partake in the reporting.
The absence of reported human disease infections after a DCD e 0.1 can be due to vector variables or the human infections did not progress into a disease. Mosquitoes, which are vectors of the virus, might have been previously eliminated by mosquito control procedures. It is also possible that the mosquitoes did not feed on humans but instead fed on other animals. Another reason is not all viral infections lead to a clinical disease. Other infections only manifest as mild forms which are often undetected. Various factors like the high capability of the human hosts immune system can play a role in disease development.
Real time reporting systems like DCD index have great potential for the management of infectious diseases outbreaks. The establishment of real time reporting systems for other diseases can be of great significance to the health industry.  Given the time before the disease is transferred from the animal host to humans, various preventive and management measures can be prepared to minimize or eliminate the occurrence of infectious disease outbreaks in humans.