22 August 2012

Microbes:  Enemy and Friend

 - Nowhere is biologic diversity demonstrated more dramatically than by microorganisms. Clearly by now, we have observed how viruses, bacteria and other microorganisms can upset the balance of our ecosystem.  Our initial image of breakouts is that these minute organisms are nothing but trouble, and, not only that, they do it big time. We see that they can easily evolve and adapt to our own defense mechanisms, then strike in an insidious manner. Right when we think it’s over, they mutate and defeat us in a couple of ways. But first, let’s review the basic differences between a bacterium and a virus. 

 - To be fair, there are species of microorganisms that serve a useful purpose. One example is Bti (Bacillus thuringiensis serovar israelensis), a gram-positive soil bacterium that produces crystalline inclusions with a highly specific insecticidal activity. It produces toxins which are effective in killing various species of mosquitoes, fungus gnats, and blackflies, while having almost no effect on other organisms. Bti strains possess the pBtoxis plasmid which encodes numerous Cry and Cyt toxins, including Cry4, Cry10, Cry11, Cyt1 and Cyt2. They have been used as a larvicide for many years and is now becoming widely available for you to buy and use yourself at home.  - Formulations include small, donut-shaped  form "Mosquito Dunks"/"Mosquito Bits". It is also available in bulk liquid or granular formulations for commercial and public agency use. A home gardener’s spray,“GET OFF ME! Natural Bug Spray”, is also available. These Bti solutions can be produced in small batches making them practical for local area mosquito control. Another natural solution is made from efficacious isolates of Bacillus sphaericus Neide. B. sphaericus has been shown to persist longer than Bti in polluted habitats and under certain circumstances. A disadvantage of B. sphaericus has been the development of resistance in certain populations of Culex quinquefasciatus Say and Culex pipiens Linnaeus. 

- The factors for their larvicidal action depend on the nature of the breeding potential of the environment, type of mosquito, their respective feeding strategies, rate of ingestion, age and density of larvae, habitat factors (temperature, solar radiation, depth of water, turbidity, tannin and organic content, presence of vegetation, etc.), and the different aspects  of the formulations.  Let us remember though, that the best way to control mosquitoes is still to get rid of standing water on your property. Prevention and control begin with understanding disease transmission and insect vector life cycle. This is probably the most neglected aspect in the control of disease. The seasonal pattern of WNV parallels the proliferation (life cycle) of the insect. This makes this quite relevant to our current West Nile Virus-mosquito epidemic connection.

Back to Bacterial Basics

 - Eukaryotes are organisms containing a membrane-bound nucleus. Among the groups of eukaryotic microorganisms are the algae, the protozoa, the fungi, and the slime molds. On the other hand, prokaryotes are organisms in which DNA is not physically separated from the cytoplasm. Eukaryotes and prokaryotes are organisms because they contain all of the enzymes required for their replication. Furthermore, they possess the biologic equipment necessary for the production of metabolic energy.  Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, and have a wide range of shapes, ranging from spheres to rods and spirals. They thrive in soil, plants, water, as well as in the gut flora and integument. 

Gram Positive Bacterium
A diagram of a typical prokaryotic cell.
March 2008; Author Mariana Ruiz Villarreal 

 - There are approximately ten times as many bacterial cells in the human flora as there are human cells in the body.  Bacteria display a wide diversity of shapes and sizes.  Most bacterial species are either spherical (cocci), or elongated (bacilli). They are broadly classified as Gram positive (no outer membrane), and Gram negative (outer membrane present). Gram stain primarily detects peptidoglycan, which is present in a thick layer in Gram positive bacteria. They exist simply as single cells, others associate in characteristic patterns. Neisseria, (e.g. gonococcus), form diploids (pairs).  They have a cell wall and self-preserving intracellular structures, responsible for the respiration and metabolism of the organism. The bacteria’s outer lipid membrane, or cell membrane, encloses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. Biochemical reactions, such as energy generation, occur by concentration gradients across membranes. Internally, there is a lack of membranes  which means reactions such as electron transport occur across the cell membrane between the cytoplasm and the periplasmic space (region bordered by two selective permeable barriers, i.e. biological membranes, which are the inner membrane (i.e. cytoplasmic membrane) and the outer membrane in Gram-negative bacteria). They may need oxygen (aerobic bacteria), or not (anaerobic bacteria). They thrive on extracellular ions and the like, and do not need a host to give it the ability to multiply. 

Going Viral

- In 1892, Dimitri Ivanovsky described a non-bacterial pathogen infecting tobacco plants. Then in 1898 Martinus Beijerinck eventually identified the tobacco mosaic virus. a positive-sense single stranded RNA virus that infects plants, especially tobacco and other members of the family Solanaceae, known as the nightshade or potato family. Since that time, at least 5000 viruses have been described, quite a few of which still have to be properly studied in detail. The unique properties of viruses set them apart from living creatures. 

Electron microscopy of West Nile virus, taken from lab, 
rights retained to displayed author.
20 November 2007 (first version); 10 June 2010 (last version)
 Original uploader was PhD Dre

[Magnification in a Scanning Electron Microscope  

 - There are seven classes of viruses. (The term virus is from the Latin, virulentus, meaning poisonous.) Viruses are not cells, but rather particles consisting of 2 to 3 things, i.e. genetic material made from DNA or RNA, the protein coat that protects these genetic materials, and in a few cases a lipid envelope that that surrounds the protein coat when the virus is not inside a host cell. The virus is of an extremely smaller size, at 1/100 the size of the usual bacterium. Viruses can be shaped as a helix, or can be icosahedral (20 identical equilateral triangular faces, 30 edges and 12 vertices), and in some still more complex geometries. Of course, because of its minute size, they cannot be seen using the optical microscope commonly found in labs.  They can be visualized usually through special imaging techniques such an electron microscope such as a SEM (scanning electron microscope) which can render a magnification over a range of up to 6 orders of magnitude from about 10 to 500,000 times. Polymerase Chain Reaction (PCR) is another method for studying bacteria. This technique enhances (amplifies) the DNA sequence wherein a short region of the DNA (fragments containing a few copies) is amplified up to ~10 kilo base pairs (kb). The underlying mechanism for PCR is called thermal cycling.

 - The range of host cells that a virus can infect is called its "host range". This is how viruses can be transmitted from plant to plant, plant to arthropod, arthropod to animal, or man, and this is carried out by a vector like an insect feeding on the infected plant’s nutrients. Virus transmission is also often times effected through droplets such as sneezing, and that is exemplified by respiratory viral illnesses. Still others are transmitted through blood products. Viruses lack many of the attributes of cells, including the ability to replicate.

 - Viruses are obligatory parasites, meaning they need a host cell to invade and where they then hijack the host cell’s genetic material. There the virus redirects the host's enzymatic machinery to functions associated with replication of the virus. As they infiltrate the host’s immune cells, they have the ability to knock out the immune system resulting in severe and long-term disease. Viral infections however, are also capable of inducing immunity of the host. This way, the host’s immune system protects against future infection. A number of transmissible plant diseases are caused by viroids, which are small, single-stranded, covalently closed circular RNA molecules existing as highly base-paired rod-like structures; they do not possess capsids.  The extracellular form of the viroid is naked RNA—there is no capsid of any kind. The RNA molecule contains no protein-encoding genes, and the viroid is therefore totally dependent on host functions for its replication. 

 - Antibiotics have no effect on viruses, unlike bacteria that are usually eliminated by this drug class by different mechanisms. It is asked, where and when did viruses begin to appear? Are they primitive genetic molecules that have evolved into infectious agents? One might ask, “Were they remnants of decaying plant or animal tissue, remnant particles that became a rogue part of the environment, very much like plants and animals?” One thing seems sure, and that is, viruses need the host to exist, multiply, and thrive. Recent developments involve using viruses as vectors for delivery of therapeutic substances.
The Virus-Mosquito-Bacteria Circle

  - In the current West Nile Virus outbreak, we can see how knitted together are the different infectious elements and their targets. First, the mosquito bites an infected animal, a vertebrate such as birds, humans, horses, alligators and others, which then serves as a host. The mosquito can also acquire the virus from an infected animal. The mosquito then lays its eggs in standing water. Some lay eggs near the water's edge; others attach their eggs to aquatic plants, where it goes through its different stages: egg, larva, pupa, and adult or imago . Thus, as the juvenile forms mature, they become vectors - multiplied exponentially. Enter now the bacteria, Bti (Bacillus thuringiensis serovar israelensis) which kill off the mosquito larvae. This in turn markedly reduces the numbers of the vector. As we apply the Bti, we are using the mosquito larva’s natural enemy to control our own natural enemy, the mosquito with West Nile Virus. In other words: bacteria controls virus, indirectly. While Bti may help in the control of mosquito populations, its persistence in the environment may raise a few questions. Anything in inordinate numbers can spell imbalance in an already threatened ecosystem. Controlling the buildup of stagnant water is still the best way of controlling the multiplication of mosquitoes. 
 - On a lighter note, horticulturists as well as ornamental plant growers can both use Bti to control mosquitoes that may thrive in irrigation water barrels and pails. In a big plot of land, good drainage should be encouraged thereby limiting the mosquitoes and the use of any control agent. Those who own dipping or swimming pools should keep them clean with the appropriate disinfectants (e.g. sodium hypochlorite and copper sulfate).

Larvae of an Anopheles sp., about 8 mm long. Found in a pond in Munich, Germany, in June 2007. Typical are the greenish color and a resting position parallel to the water surface.
17 June 2007
by Steffen Dietzel

 - As far as the Preventive Medicine principles are concerned, the measures used to control the West Nile Virus carriers are also  measures for other mosquito-borne diseases.

For U.S. West Nile Virus outbreak demographics, visit this page for updates

- As far as August 14, there appears to be no new case of WNV. This is good. At least the proper surveillance and control methods are paying off, albeit a tad slow at first.Total number of cases as of  Aug. 14, 2012 is 693. Since 1999, more than 30,000 people in the United States have been reported as getting sick with West Nile virus. 

 - In the meantime, awareness and surveillance continues so as not to miss the patient with atypical clinical presentation. Travelers need to be cautious especially  in going to areas with swamps, stagnant rivers, and other areas where their is an outbreak. So it would be wise to consult the map as noted in aforementioned page

 - As newer technology and better preventive methodology develops, it is hoped that the big brothers in healthcare, eg., WHO and CDC, will be able to prepare us in any eventuality. In this outbreak alone, we have seen the melding of clinical acumen, public health/preventive medicine and various areas of bioinformatics. Sometimes, in all the hustle of the situation, we tend to overlook the role of the computer scientist and physician in their untiring efforts to keep us healthy.

 - Keep smart, keep safe. Til another day...

 - Fernando Yaakov Lalana, M.D. 

 - P.S. If you would like to see some interesting Scanning Electron Microscope images do visit this page.

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