As this year’s flu season gets underway in the northern hemisphere, new research finds that when it comes to flu vaccination, more appears to be better. Two new studies published in the open access journal PLoS Medicine show that increasing the number of people vaccinated against influenza can decrease the burden of the disease, and not just in the individuals receiving the vaccine.
Targeted vaccination programs, in which flu vaccine is recommended for particular groups at high risk of spreading or experiencing complications of influenza, are commonly implemented. In contrast, the Canadian province of Ontario initiated a universal immunization program in 2000, in which flu vaccination is promoted and provided free of charge to everyone over the age of 6 months. The first study evaluated the effect of this universal immunization program on influenza-associated health outcomes. The researchers analyzed national and provincial data from 1997 to 2004, to compare changes in Ontario’s flu outcomes before and after introduction of universal vaccination with outcomes in other provinces, which continued targeted vaccination programs. They found that, compared with other Canadian provinces, Ontario’s universal vaccination program was associated with reductions in influenza outcomes including flu-related deaths, hospitalizations, and visits to emergency departments and doctors’ offices. The results did suggest, however, that increasing immunization rates may not be as effective in reducing mortality and health care use in older people, particularly those over 75 years of age, compared to younger people. However, even with enhanced access to free flu vaccines in Ontario, only an estimated average of 38% of the overall household population reported receiving them, suggesting that protection of older people by higher immunization rates of younger contacts who might expose them to influenza may still be of benefit.
The second study further investigated the concept of herd immunity, by which immunization of some individuals protects the overall population by reducing exposure of those who are not immunized. Using a mathematical model to simulate spread of influenza in nursing homes, researchers found that increasing the number of health care staff who are vaccinated can protect additional patients from influenza. They calculated that increasing the proportion of vaccinated health care workers from zero to 100% in a 30-bed nursing home department would reduce patient infections by about 60%, and that vaccinating seven health care workers would on average prevent one patient from getting influenza. They also found that no level of health care worker vaccination guarantees complete herd immunity, suggesting that even at high levels of immunization, increasing the number of nursing home staff who are vaccinated against flu each year will further reduce risk to patients. The authors also note that random variation, which occasionally leads to large outbreaks, limits the ability of small vaccination trials to assess the actual relationship between health-care worker vaccination and patient risk of influenza.
Montage of high-speed video clips showing sporangiophore discharge in the fungus Pilobolus kleinii. The videos were obtained at camera frame rates of up to 250,000 fps. Each discharge is completed in less than 0.25 milliseconds; an eye blink takes 100 milliseconds, or 400 times longer! The music is Verdi’s Anvil Chorus. Credit: Yafetto et al
Microscopic coprophilous (dung-loving) fungi help make our planet habitable by degrading the billions of tons of faeces produced by herbivores. But the fungi have a problem: survival depends upon the consumption of their spores by herbivores and few animals will graze on grass next to their own dung. Evolution has overcome this obstacle by producing an array of mechanisms of spore discharge whose elegance transforms a cow pie into a circus of microscopic catapults, trampolines, and squirt guns. A new paper examines the operation of squirt guns that fire spores over distances of more than 2 metres. The researchers used high speed cameras running at up to 250,000 frames per second to capture these blisteringly fast movements. Spores are launched at maximum speeds of 25 meters per second impressive for a microscopic cell corresponding to accelerations of 180,000 g. In terms of acceleration, these are the fastest flights in nature. The paper is significant for a number of reasons. This is the first study utilizing ultra-high-speed video cameras to capture the events of spore discharge in ascomycete and zygomycete fungi. Previous investigators relied upon models to predict ballistic parameters and produced erroneous estimates of velocities and accelerations. These estimates were then used to suggest that pressures within the spore guns were very high. Fungal cells generate pressure by osmosis and the authors used a combination of spectroscopic methods to identify the chemical compounds responsible for driving water influx into the guns. These experiments showed that the discharge mechanisms in fungi are powered by the same levels of pressure that are characteristic of the cells that make up the feeding colonies of fungi. Therefore, the long flights enjoyed by spores result not from unusually high pressure, but from the way in which explosive pressure loss is linked to the propulsion of the spores. There are similarities between the escape of the spores and the expulsion of ink droplets through nozzles on inkjet printers. Another important aspect of the new work is the way that it has allowed the researchers to test different models for the effect of viscous drag on microscopic particles and identify limitations in previous approaches to modeling. This information is very important for future biophysical studies on spore and pollen movement, which have implications for the fields of plant disease control, terrestrial ecology, indoor air quality, atmospheric sciences, veterinary medicine, and biomimetics.
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The Fastest Flights in Nature: High-Speed Spore Discharge Mechanisms among Fungi. 2008 PLoS ONE 3(9): e3237
Background: A variety of spore discharge processes have evolved among the fungi. Those with the longest ranges are powered by hydrostatic pressure and include “squirt guns” that are most common in the Ascomycota and Zygomycota. In these fungi, fluid-filled stalks that support single spores or spore-filled sporangia, or cells called asci that contain multiple spores, are pressurized by osmosis. Because spores are discharged at such high speeds, most of the information on launch processes from previous studies has been inferred from mathematical models and is subject to a number of errors.
Methodology/Principal Findings: In this study, we have used ultra-high-speed video cameras running at maximum frame rates of 250,000 fps to analyze the entire launch process in four species of fungi that grow on the dung of herbivores. For the first time we have direct measurements of launch speeds and empirical estimates of acceleration in these fungi. Launch speeds ranged from 2 to 25 meters per second and corresponding accelerations of 20,000 to 180,000 g propelled spores over distances of up to 2.5 meters. In addition, quantitative spectroscopic methods were used to identify the organic and inorganic osmolytes responsible for generating the turgor pressures that drive spore discharge.
Conclusions/Significance: The new video data allowed us to test different models for the effect of viscous drag and identify errors in the previous approaches to modeling spore motion. The spectroscopic data show that high speed spore discharge mechanisms in fungi are powered by the same levels of turgor pressure that are characteristic of fungal hyphae and do not require any special mechanisms of osmolyte accumulation.
Trichomonas vaginalis is a flagellated protozoan parasite, 10-30 µm in diameter, and is responsible for one of the most widespread sexually transmitted diseases worldwide, trichomoniasis or “trich”. The WHO has estimated that 180 million infections are acquired annually worldwide. The main signs of a Trichomonas infection in women are abdominal pain, itching, and presence of a foul-smelling discharge with abundant leukocytes, while in men the infection is mostly asymptomatic, although it can sometimes lead to urethritis, prostatitis, and epididymitis. Infection with this organism is also associated with severe complications, such as infertility and enhanced predisposition to cervical tumours.
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Diagnosis depends on finding trophozoites in secretions of the genital tract from men or women. In cases where the numbers of organisms are very low, the trophozoites can be cultured to increase their numbers. There is no cyst in the parasite life cycle, so transmission occurs via the trophozoite stage. These motile cells have four flagella (visible in this video if you look carefully) and single nucleus. There is also a median rod called the axostyle which is characteristic of the trichomonads. However, this is not clearly visible without staining the cells.
The pathogenesis of Trichomonas infections occurs through cytopathogenicity of the organism for vaginal epithelial cells. Adhesion of the parasite to the target cell is essential for the maintenance of infection and for cytopathogenicity. Flattened, adherent forms can be seen in the last two clips in this video.
X-ray crystallography has been an important technique for biologists since the 1940s and 50s, revealing the three-dimensional structure of complex biological molecules such as haemoglobin, DNA and viruses. But the technique has a severe limitation: it only works with molecules that form into crystals and that turns out to be a tiny fraction of the proteins that make up living things. A team of US researchers have created the first image of a single uncrystallized virus particle using x-ray diffraction.
The virus they studied was murine herpesvirus-68 (MHV-68). A herpes virion has an asymmetric tegument and envelope outside of the icosahedrally symmetric capsid composed of defined numbers of subunits. However, each virion may have a different size of tegument and envelope, and the virus capsid is not necessarily in the center of the virion. While cryo-electron microscopy can determine the capsid structure of herpesviruses by averaging over thousands of virus particles, the reconstructions of pleomorphic virions obtained by cryo-electron tomography are limited in low image contrast and high levels of noise.
The trick with the new technique is to take a diffraction pattern of the virus and then subtract the diffraction pattern of its surroundings. This breakthrough paves the way for scientists to start teasing apart the three-dimensional structures of the many viruses and proteins that have eluded biologists to date.
Quantitative Imaging of Single, Unstained Viruses with Coherent X-rays. 2008 arXiv:0806.2875v1
Since Perutz, Kendrew and colleagues unveiled the structure of hemoglobin and myoglobin based on X-ray diffraction analysis in the 1950s, X-ray crystallography has become the primary methodology used to determine the 3D structure of macromolecules. However, biological specimens such as cells, organelles, viruses and many important macromolecules are difficult or impossible to crystallize, and hence their structures are not accessible by crystallography. Here we report, for the first time, the recording and reconstruction of X-ray diffraction patterns from single, unstained viruses. The structure of the viral capsid inside a virion was visualized. This work opens the door for quantitative X-ray imaging of a broad range of specimens from protein machineries, viruses and organelles to whole cells. Moreover, our experiment is directly transferable to the use of X-ray free electron lasers, and represents a major experimental milestone towards the X-ray imaging of single macromolecules.
Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host. PLoS Pathog 2008 4(6): e1000090
Pathogenic spirochetes are bacteria that cause a number of emerging diseases worldwide, including Lyme disease. Spirochetes exhibit an unusual form of helical motility and can infect many different tissues. However, the mechanism by which they disseminate from the blood to target sites is unknown. Direct visualization of bacterial pathogens at the single cell level in living hosts is important, since this approach is likely to yield critical insight into disease processes. In a recent paper, researchers engineered a fluorescent strain of Borrelia burgdorferi, the Lyme disease pathogen, and used confocal microscopy to directly visualize these bacteria in real time and in 3D in living mice. They found that spirochete interaction with and dissemination out of the vasculature was a multi-stage process of unexpected complexity and that spirochete movement appeared to play an integral role in dissemination. This is the first report of high resolution 3D visualization of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.
In the first section of this video you can see B. burgdorferi moving in the ear of a living mouse. The second section shows B. burgdorferi in a postcapillary venule in the skin of the mouse, and the third section shows the actual moment of escape from the blood vessel into the surrounding tissue.
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Many but not all bacteria exhibit motility, i.e. self-propelled motion, under appropriate circumstances. Motion can be achieved by one of three mechanisms.
Most motile bacteria move by the use of flagella, rigid structures 20 nm in diameter and 15-20 µm long which protrude from the cell surface, e.g. the Chromatium cells in the video. In some bacteria, there is only a single flagellum – such cells are called monotrichous. In these circumstances, the flagellum is usually located at one end of the cell (polar). Some bacteria have a single flagellum at both ends – amphitrichous. However, many bacteria have numerous flagella; if these are located as a tuft at one end of the cell, this is described as lophotrichous (e.g. Chromatium), if they are distributed all over the cell, as peritrichous.
Flagella consist of a hollow, rigid cylinder composed of a protein called flagellin, which forms a filament anchored to the cell by a curved structure called the hook, which is attached to the basal body. Flagellae are, in effect, rotary motors comprising a number of proteinaceous rings embedded in the cell wall. These molecular motors are powered by the phosphorylation cascade responsible for generating energy within the cell. In action, the filament rotates at speeds from 200 to more than 1,000 revolutions per second, driving the rotation of the flagellum. The organization of these structures is quite different from that of eukaryotic flagella. The direction of rotation determines the movement of the cell. Periodically the direction of rotation is briefly reversed, causing what is known as a “tumble”, and results in reorientation of the cell. When anticlockwise rotation is resumed, the cell moves off in a new direction. Watch for the tumbles in this video. This allows bacteria to change direction. Bacteria can sense nutrients and move towards them – a process is known as chemotaxis. Additionally, they can also move away from harmful substances such as waste products and in response to temperature, light, gravity, etc. This apparently intelligent behavior is achieved by changes in the frequency of tumbles. When moving towards a favourable stimulus or away from an unfavourable one, the frequency of tumbles is low, thus the cells moves towards or away from the stimulus as appropriate. However, when swimming towards an unfavourable or away from a favourable stimulus, the frequency of tumbles increases, allowing the cell to reorient itself and move to a more suitable growth.
The second type of motility is shown by Spirochaetes, helical bacteria which have a specialized internal structure known as the axial filament which is responsible for rotation of the cell in a spiral fashion and consequent locomotion. The video shows highly motile Rhodospirillum rubrum cells. Watch the corkscrew motion of the cells through the medium.
The third mechanism is gliding motility. Gliding motility is the movement of cells over surfaces without the aid of flagella, a trait common to many bacteria. Gliding bacteria all secrete copious slime, but the exact mechanism which propels the cells is not known. The gliding motility apparatus which propels the cells involves a complex of proteins, yet the full nature of this “motor” and how the components interact is not understood. You can watch an Oscillatoria cell gliding in real time in the video.
However, beware for not everything that moves is motile! Under the microscope, motile bacteria seem to move in a purposeful way, though they may frequently change direction. However, even dead cells, such as those in this video, move. Rapid movement is due to capillary action or convection currents on the microscope slide. However, the motion which causes most problems is Brownian motion, first observed in 1827 by the English botanist Robert Brown. This is due to random molecular bombardment of tiny bacterial cells by the molecules of the solvent. A microbiologist needs to learn to distinguish the effects of Brownian motion from true bacterial motility.
Humans are hosts to nearly 300 species of parasitic worms and over 70 species of protozoa, some derived from our primate ancestors and some acquired from the animals we have domesticated or come into contact with during our history (History of human parasitology. Clin Microbiol Rev 2002 15: 595-612). The best-documented parasitic disease known from ancient times is caused by the nematode worm Dracunculus medinensis. The earliest description is from an Egyptian papyrus from 1500 BC that refers to both the nature of the infection and to techniques for removing the worm. Confirmation of the presence of this worm in ancient Egypt comes from the finding of a well-preserved worms in Egyptian mummies. Dracunculiasis, or Guinea worm disease, is one of the few diseases unambiguously described in the Bible, and most parasitologists accept that the “fiery serpents” that struck down the Israelites in the region of the Red Sea after the Exodus from Egypt somewhere between 1250 to 1200 BC were actually Guinea worms.
The adult worms live in the subcutaneous connective tissues of their victims, from which the females emerge to release thousands of larvae into water, where they are taken up by intermediate hosts, tiny aquatic crustaceans called Cyclops. In these hosts they mature into infectious larvae that infect humans when the crustaceans are accidentally swallowed in contaminated drinking water. On maturity, the large female worm, up to nearly a metre in length, protrudes from the skin, usually of the leg, and causes intense inflammation and irritation. The effects of the disease are crippling. Its victims develop large ulcers, usually in the lower leg. The ulcers swell, sometimes to the size of a tennis ball, and burst, releasing the spaghetti-like parasitic worm. Victims experience a pain so excruciating that they say it feels as if their leg is on fire. The searing pain compels people to jump into water, often the community’s only source of drinking water, to relieve the pain. When the infected person immerses his or her leg in the water, the worm in the leg releases thousands of larvae. The larvae are then ingested by Cyclops that live in the water. Thus the cycle begins again – when people drink the water, they are in effect drinking in the disease.
The most common way to treat Guinea worm disease involves wrapping the worm around a stick. This treatment has been employed for millennia and may have inspired the Rod of Asclepius which historically has symbolized the medical profession. As the adult worm begins to emerge from the patient’s skin, it is wound around a stick, then further extracted by a few centimeters per day. This slow process can take days or even weeks, but it is required to avoid breakage and leaving behind a portion of the worm. Leaving a portion of the dead worm remain within the host’s body increases the risk of infection, and can trigger immune responses resulting in pain and swelling. In many countries, a broken worm is immediately removed surgically, or the worm can be excised surgically from the very beginning if health care facilities are available. Antihelminthic drugs such as metronidazole or thiabendazole are sometimes used in conjunction with physical extraction. However, one study found that antihelminthic therapy was associated with aberrant migration of worms, resulting in infection in areas other than the lower extremity.
Dracunculiasis is a classic example of a neglected tropical disease, a symptom of poverty and disadvantage. Those most affected are the poorest populations often living in remote, rural areas, urban slums or in conflict zones. With little political voice, neglected tropical diseases have a low profile and status in public health priorities. In 1997 the World Health Assembly pledged to completely eradicate Guinea worm disease. This is no small task, but there are several factors which make eradication a possibility. Dracunculiasis is the first parasitic disease targeted for eradication because:
Diagnosis is easy and unambiguous (presence of an emerging adult worm).
The transmission agent, Cyclops, is not a mobile vector as is a mosquito.
The incubation period in both Cyclops and humans is of limited duration.
Interventions are effective, low cost, and relatively simple to implement.
The disease has a limited geographic distribution and is seasonal in nature.
Success in eliminating the disease has been demonstrated in several countries in Asia and the Middle East.
There is no known animal reservoir.
Is Dracunculiasis eradication close? In 2007 the WHO announced that Guinea worm disease now affects around 25,000 people in nine countries, compared with an estimated 3 million people were infected in over 20 countries in the early 1980s. Twelve countries were declared Guinea worm-free in early March. If progress continues at this rate, the disease could be eradicated in less than two years. It is probable that complete eradication will take quite a few years yet, although it should be possible to eliminate the disease from seven countries in a couple of years, leaving only two endemic countries, Sudan and Ghana (Dracunculiasis eradication by 2009: will endemic countries meet the target? Tropical Medicine & International Health 2007 12: 1403-1408). One lesson to be drawn from the problems of local ownership and the experience of cash rewards is that there are dangers in throwing money at the problem. While the eradication initiative badly needs additional resources, it needs them at such a level and managed in such a way that they do not distort the priorities of the health care system, or exceed the capacity of local staff to manage them. The amounts needed are not large, but their continuity and flexibility is important. Given the highly seasonal transmission of dracunculiasis, the resources must be available at very specific times of the year, which is not always achieved. In spite of the difficulties, complete worldwide eradication of this ancient disease is drawing nearer.
The structure of all retroviruses is similar, although there are some minor differences. Virus particles are far too small to see, the closest we can come to are electron micrographs. To make transmission electron micrographs, the specimen (containing virus particles) are fixed and stained with a metal-containing dye. The more dye different areas of the specimen take up, the darker they appear in the electron micrograph.
In the centre of an HIV particle, there are two molecules of RNA which together make up the genome of the virus. Associated with the RNA are two enzymes, reverse transcriptase and integrase. The genome is enclosed in a conical core consisting of the nucleocapsid proteins. Outside this is an icosahedral protein capsid, which in turn is enclosed by the matrix protein layer. The whole particle is surrounded by a lipid bilayer known as the virus envelope. The transmembrane protein penetrates through the envelope and anchors the surface glycoprotein on the outside of the particle.
To see more detail in virus particles, special imaging techniques are needed. Cryo-electron tomography makes a three dimensional reconstruction from a series of two dimensional transmission electron micrographs taken at extremely low temperatures in order to preserve the structure of the particle. The individual micrographs represent slices though the virus particle which are put together on a computer to construct a three dimensional representation with false colours added for additional clarity.
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The Chlamydia are a genus of obligate intracellular bacteria. There are only a few species in this genus and Chlamydia trachomatis is the one which causes sexually transmitted infections in humans. Chlamydia is the most common sexually transmitted infection in the UK and probably worldwide. In developing countries, Chlamydia infection of the eye is the most common cause of preventable blindness. The UK national screening programme has found that 10% of both men and women aged 18 to 25 carry the bacterium, and the Health Protection Agency says that cases of Chlamydia infection have increased by more than 200 per cent in England in the past decade.
Chlamydia infections of the genital tract frequently show no symptoms at all, particularly in women, and so may go untreated for years. For a long time, chronic infection has been known to harm fertility in infected women, but it has recently been shown that infected men also have decreased fertility. This was demonstrated when a team of doctors from Mexico examined sperm from men infected with Chlamydia who had failed to father a child. Using a microscopic analysis technique, they found the level of DNA fragmentation in their sperm was more than three times higher than in healthy men. The concentration of their sperm, and its ability to swim were also poor, and there were increased levels of defects in sperm shape. The researchers then treated 95 of the infertile men with antibiotics effective against Chlamydia and found the DNA sperm damage improved by an average of 36% after four months. During that period, 13% of the couples became pregnant but after the treatment was finished, 86% achieved a pregnancy.On the BBC website, Dr Allan Pacey, Secretary of the British Fertility Society, is quoted as saying that more needs to be done to target the younger generation.
The message is that we might think of Chlamydia as a disease that damages female fertility, but we need to think again. Chlamydia is getting out of control. We have got to encourage men as well as women to go for screening, but men are more reluctant to do this if they don’t have symptoms. It is the 18 to 25 age group that is of most concern. There should be a page on Facebook you can log onto and sort screening out.
Unlike some of the sexually-transmitted diseases discussed on MicrobiologyBytes (e.g. HIV infection, which is a pretty good reason not to have unprotected sex), Chlamydia infections are easy to treat with a short course of antibiotics – if they can be diagnosed. Because of the unusual intracellular lifestyle of the bacterium, conventional microbiological culture methods are of limited use in detecting the organism. In recent years, techniques involving DNA amplification have become the mainstream diagnostic method.
As this year’s flu season gets underway in the northern hemisphere, new research finds that when it comes to flu vaccination, more appears to be better. Two new studies published in the open access journal PLoS Medicine show that increasing the number of people vaccinated against influenza can decrease the burden of the disease, and not just in the individuals receiving the vaccine.
