Thursday, December 18, 2014

Collapsing Colonies and Crithidia bombi

By Matthew Lowe

Over the past decade there has been a growing concern over the phenomenon known as Colony Collapse Disorder (CCD) among commercially raised and wild bees. CCD was initially characterized in 2006 by David Hackenberg, a prolific apiarist with multiple hives in Florida and Pennsylvania1. Upon investigation of lagging production, it was discovered that despite there being no adult worker bees in 20-30% of his hives the queens and brood appeared healthy1. The loss of worker bees and production from the hive while still maintaining a seemingly healthy brood and queen has now become the definitive end result of CCD.

C. bombi
Since 2006, there have been numerous investigations into potential causes but so far no single cause has been definitively linked to CCD. Some of the more extreme claims include cell phone radiation and commercial use of insecticides, however there is no strong evidence to support that either is the cause of CCD1.  Of the potential rational causes for CCD, the most likely is an increased prevalence of parasites and viruses within the bee population1. Crithidia bombi, a recently implicated parasite, is one of the more interesting because of its ability to infect a large variety of different bee species, including those found within the pyrobombus, thoracobombus and bombus sensu stricto subgenera2.  Of the North American species infected, Occidentalis and Pensylvanicus are currently undergoing massive population decline due to CCD2. C. bombi infection is also 6 fold higher in CCD colonies as opposed to hives not experiencing CCD3.

Figure 1 from here
C. bombi is a pathogenic unicellular eukaryote with two distinct life phases; the flagellated choanomastigote and the anchored amastigote cells4. The amastigote cells upon ingestion will extend their flagella and swim as a choanomastigote until they can attach to the bee’s intestinal wall and once again become an amastigote4. Upon attachment the cell can siphon off nutrients that pass by and will divide into new amastigote cells4. Some of these cells will be excreted in feces and can be transmitted from bee to bee through the ingestion of infectious cells to begin the cycle anew2, 4, 5. The amastigote cells can either be ingested within the hive, leading to a high level of infection amongst the workers and queen, or at flowers allowing for the parasitic colonization of new hives5. Once within a hive, infection spreads rapidly until about 80% of the colony is infected6. Due to C. bombi having a genotype-genotype model of infection, in which the unique genetic profile of the host and the parasite both play a role in infection success, the highly related individuals within a hive are much more frequently infected7. However, once a cross hive infection is established it will rapidly spread within the new colony7. While infection has not been linked to any significant lethality in otherwise healthy bees, an infected bee experiencing starvation has a 50% increase in mortality5. Upon infection the bee will have a harder time distinguishing between the flowers that are the most rewarding based on color, as shown in figure 18. This suboptimal foraging will lead to less food collection for the hive as a whole and a potential minor starvation event that would cause increased mortality in the infected bees8. If a queen is infected she will have reduced ovarian capacity leading to a decreased worker population6. An infected queen also experiences a decrease in the ability to store energy as fat for the hibernation over winter greatly decreasing her chances of survival6. If the queen manages to survive the winter, she will produce fewer offspring that will also become infected6. This generational transmission pattern is particularly vexing for apiarists because an infected queen not show symptoms until the following year when she establishes a colony.  During the time between infection and diagnosis, C. bombi is also being spread to nearby flowers and potentially other hives which could account for the high infection rate in commercial colonies every year.

Figure 2 from here
This continual transmission and difficulty of removing C. bombi begs the question, why should we care if all of the bees die? The economic impact of the insect pollination industry is valued at over 150 billion euros9. As shown in figure 2, this is roughly equivalent to 10% the total value of global agricultural production9. The use of bees makes up over 70% of the insect pollination industry and the value of crops that are been pollinated is roughly 5 times higher than those that are not9. This is not even considering that the apiary industry as a whole, including all of the production facilities to manufacture bee related goods, employs millions of workers worldwide. A shrinking of the bee population will also lead to an increase in the cost of pollination services which will get passed on to the consumer as a wide variety of fruits, nuts and vegetables increase in price to compensate.

While the apiary industry is one of the main driving forces for CCD research, the disorder is not limited to just commercial hives. Commercial bees are commonly raised in greenhouses with a crop to pollinate and it has been shown that a few bees escape and carry infection into the wild10. The close confines of the greenhouse and the fact that the bees are likely more related due to being commercially raised, infection spreads rapidly between hives leading to a higher parasite load than in wild bees7, 10. Any bee that escapes can spread infectious cells out of the greenhouse and to wild bees through the shared use of nearby flowers. Due to C. bombi’s ability to infect a wide range of bee species, this could lead to collapse of wild bee colonies throughout many different regions of the world. Even more worrisome, commercial colonies are commonly transferred across the globe for pollination purposes as well as to start up new colonies1. Therefore any infection from a single colony has the potential to spread globally and infect numerous native colonies. While it has been shown that a single bee proof mesh placed over the air duct will greatly diminish the chances of escape, Commercial bee keeping still exists as a potential method of transferring infectious agents to native bee populations across the globe10. If the native population of bees were to die, thousands of native plant species would lose a prime pollinator which could lead to an inability to efficiently reproduce devastating animal populations that rely on them for food.

Though it is likely that there is no single cause to CCD, the research into it has turned up several interesting parasitic species that all are likely to play a role in colony collapse. Crithidia bombi is but one of the more interesting due to its ability to infect a wide range of bees beyond the commercial species and infection has been correlated with CCD2, 3. This in no way means that C. bombi is the sole cause of CCD but that it likely contributes to the decline of affected hives. Further research into the topic is definitely needed to determine other contributory factors that when combined will lead to CCD. Until a large scale prevention method is identified there are a few things that an individual can do to help maintain local bee populations. Becoming an apiarist and setting up a hive or two in your backyard can help provide a home for local varieties of bees and provide a strong pollination source for nearby gardens11. Planting a variety of species in gardens can also lead to more diverse food sources and increase a colony’s overall health11. By supporting further research into CCD and following through on few basic prevention methods we can hopefully save the bee population as a whole!


1.     Watanabe M. 2008. The Xerces Society » Colony Collapse Disorder: Many Suspects, No Smoking Gun. http://www.xerces.org/2008/05/01/colony-collapse-disorder-many-suspects-no-smoking-gun/
2.     Cordes N, et al. 2012. Interspecific Geographic Distribution and Variation of the Pathogens Nosema bombi and Crithidia Species in United States Bumble Bee Populations
3.     Cornman RS, Tarpy DR, Chen Y, Jeffreys L, Lopez D, et al. (2012) Pathogen Webs in Collapsing Honey Bee Colonies. PLoS ONE 7(8): e43562. doi:10.1371/journal.pone.0043562
4.     Olsen OW. 1974. Animal Parasites: Their Life Cycles and Ecology. Courier Dover Publications.
5.     Deshwai S, Mallon EB. 2014. Antimicrobial Peptides Play a Functional Role in Bumblebee Anti-trypanosome Defense. BioRxiv
6.     Erler S, Popp M, Wolf S, Lattorff HMG. 2012. Sex, horizontal transmission, and multiple hosts prevent local adaptation of Crithidia bombi, a parasite of bumblebees (Bombus spp.). Ecol Evol 2:930–940.
7.     Riddell Carolyn, et al. 2014. Insect Immune Specificity in a Host-parasite model. BioRxiv
8.     Gegear RJ, Otterstatter MC, Thomson JD. 2006. Bumble-bee foragers infected by a gut parasite have an impaired ability to utilize floral information. Proc Biol Sci 273:1073–1078.
9.     Savrieno A. 2012. Colony Collapse Disorder As Part Of An Acquisition Strategy. Seeking Alpha. http://seekingalpha.com/article/590411-colony-collapse-disorder-as-part-of-an-acquisition-strategy
10.  Otterstatter MC, Thomson JD. 2008. Does Pathogen Spillover from Commercially Reared Bumble Bees Threaten Wild Pollinators? PLoS ONE 3:e2771.
11.  What you can do about Colony Collapse Disorder - Honeybees - Silence of the Bees | Nature | PBS. http://www.pbs.org/wnet/nature/episodes/silence-of-the-bees/how-can-you-help-the-bees/36/


Trypanosoma cruzi

By Noora Hussain


The human body has an army at the ready to attack and protect itself from invaders. This army is called the immune system. It is equipped with weapons that span the physical body, are effective against many different types of attacks, and are organized in such a way that allows for strategic interactions which amplifies the body’s defenses. When a parasite wages war on the body, the body’s army goes into battle with full force. Winning results in successful elimination of the parasite from the body, but may leave the immune system weakened. Losing the battle can result in disease. Parasites have their own arsenal of weapons, which allows them to defeat the immune system. Parasites have mechanisms that shield them from host’s defenses and have ways to attack specific target cells. One such parasite that wields its own sword and shield is Trypanosoma cruzi. This motile protozoan pathogen is the causative agent of Chagas disease.1 Chagas disease affects the cardiac and digestive systems of the body and can cause acute or chronic infections2.  The parasite T. cruzi uses offensive mechanisms to counterattack a host cell’s defenses.
Chagas disease affects eight million people in Latin America.2 The disease was discovered in 1909 by the Brazilian physician Carlos Chagas, who named the parasite after his mentor Oswaldo Cruz.3 Chagas disease can cause acute or chronic infection that affects many different cells of the body.2 Clinical manifestations of acute infections include myocarditis, pericardial effusion, or meningoencephalitis.3 After the initial acute stage subsides, the diseases enters the chronic stage.3 Most patients can survive with the chronic infection, but a small percentage of patients develop cardiomyopathies within a year.3 Infection by T. cruzi occurs in a cycle and involves blood sucking insect vectors belonging to the Reduviidae family. 4 From the insect, the parasite comes into contact with a human or a wild animal.2 Then, it goes back to the insect when the insect feeds off the infected human.2 When the blood sucking insect lands on skin to feed, it also defecates in that spot leaving behind T. cruzi infected feces.5 Fecal droplets can get passed inside of humans through mucosa or through breaks in the epithelial barrier.5 T. cruzi can also infect through oral transmission with infected foods.5 Another much less common transmission mechanism is from blood transfusion.5 Congenital transmission from infected mother to child is also possible, but like blood transfusions is not a very common mechanism.5
Trypomastigotes are the infectious forms of T. cruzi and they infect the endothelial and mucosal cells of humans and other mammals.6 They invade these cells in order to differentiate and replicate inside of the host cell lysosome and cytoplasm.6 The invasion mechanism of T. cruzi is unique because it uses the host cell machinery that would generally be used against a protozoan parasite. T. cruzi trypomastigotes are highly motile7. A flagellum is attached to the cell body of T. cruzi, which enables the parasite to move on its own.8 Active motility of T. cruzi is a mechanism that the parasite uses to penetrate through the host cell membrane.9 After it gains entry, the host cell is infected.9 Once the host cell is infected, the trypomastigotes undergo cytokinesis, but their nuclei do not divide.9 The division occurs towards the back end of the basal body where the flagellum is attached.9 Through this process, the unnecessary flagellum is discharged into the host cell cytoplasm where it is then degraded.9 
The surface of T. cruzi provides a shield for the parasitic pathogen. This enables the parasite to travel throughout the body without being defenseless against the host’s immune system. The major surface components of T.cruzi provide the parasite with protection against the host’s cell defenses and enables the parasite to adhere to specific target cells for invasion.5 Mucin is a glycoprotein and one of the major surface components that plays a role in infection.1 Mucin sticks out from the outer phospholipid layer of T. cruzi’s plasma membrane.5 They are anchored  to the plasma membrane by glycosylphosphatidylinositol (GPI).1 These GPI-anchored glycoproteins cover the majority of the T. cruzi’s surface.5 Mucins recognize and target endothelial cells for invasion.5  They attach themselves onto the lipid bilayer of host cells.10 A signal is transduced that directs the glycoprotein into the cytoplasm and to the endoplasmic reticulum of the host cell.10 Once inside the host cell, T. cruzi can also interact with other organelles in the cytoplasm and use them to mediate infection.10
Host cells have lysosomes to remove unwanted material from inside of their cells. Normally, a lysosome would ingest, destroy, and secrete an invading pathogen. However, upon infection, T. cruzi’s plasma membrane fuses with the host cell lysosome, creating what is called a lysosome derived parasitophorous vacuole.6 The formation of the parasitophorous vacuole anchors the parasite to a structure of the host.6 The anchored parasite can undergo replication before disseminating into the host’s bloodstream and throughout the body.6 T. cruzi interacts with lysosomes of the  host cell because they have a low pH value.6  Having a highly acid organelle is a defense weapon of the host, but is used against the host when it facilitates trypomastigotes differentiation, replication, and dissemination.6 The parasitophorous vacuole membrane is disrupted and the acidic environment can have its full effect on the trypomastigotes.6  Disruption of the membrane is caused by the release of the pore forming molecule TcTox from trypomastigotes.6 Release of this molecule is triggered by the lysosome’s acid environment.6  Acidity also serves as a trigger to initiate differentiation of trypomastigotes into amastigotes.6 Amastigotes replicate, exit the lysosome, and disseminate into the blood stream.3 This spreads infection to other cells of the body.3-6
            The outcome of a battle between a parasite and the human body is critically important. A human’s immune system is well equipped to defend against many infections. However, parasites have developed mechanisms that provide them with a good offense and can retaliate against the immune system. So, in a battle between the two, the immune system does not always defeat the invader and the parasite can conquer and win.  

References
  1. Gonzalez MS, Souza MS, Garcia ES, Nogueira NFS, Mello CB, et al. (2013) Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus. PLoS Negl Trop Dis 7(11): e2552. doi:10.1371/journal.pntd.0002552
  2. Rassi Jr, A., Rassi, A., & Marin-Neto, J. A. (2010). Chagas disease. The Lancet, 375(9735), 17-23. doi:10.1016/S0140-6736(10)60061-X
  3. Pereira Nunes, M. C., Dones, W., Morillo, C. A., Encina, J. J., & Ribeiro, A. L. (2013). Chagas Disease. Journal of the American College of Cardiology, 62(9), 767-776. Retrieved from https://www-clinicalkey-com.ezp2.lib.umn.edu/#!/content/playContent/1-s2.0-S073510971302250X
  4. Prata, A. (2001). Clinical and epidemiological aspects of Chagas disease. Lancet Infectious Diseases, 1(2), 91-100. doi:10.1016/S1473-3099(01)00065-2
  5. Campo, V. A., Frasch, A. C., Buscaglia, C. A., & Noia, J. M. (2006). Trypanosoma cruzi surface mucins: host-dependent coat diversity. Nature Reviews Microbiology, 4, 229-236. doi:10.1038/nrmicro1351
  6. Burleigh, B. A. and Woolsey, A. M. (2002), Cell signalling and Trypanosoma cruzi invasion. Cellular Microbiology, 4: 701–711. doi: 10.1046/j.1462-5822.2002.00226.x
  7. Andrade, L. O., & Andrews, N. W. (2005). The Trypanosoma cruzi–host-cell interplay: location, invasion, retention. Nature Reviews Microbiology, 3, 819-823. doi:10.1038/nrmicro1249
  8. Sacks, D. (2014). Lost but Not Forgotten. Cell Host & Microbe, 16(4), 423-425. doi:10.1016/j.chom.2014.09.017
  9. Kurup, S. P., & Tarleton, R. L. (2014). The Trypanosoma cruzi Flagellum Is Discarded via Asymmetric Cell Division following Invasion and Provides Early Targets for Protective CD8+ T Cells. Cell Host & Microbe, 16(4), 439-449. doi:10.1016/j.chom.2014.09.003
  10. Canepa, G. E., Mesias, A. C., Yu, H., Chen, X., & Buscaglia, C. A. (2012). Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins. The Journal of Biochemistry, 287, 26365-26376. doi: 10.1074/jbc.M112.354696

Tuesday, December 16, 2014

Bow chicka bovine: sexually transmitted Tritrichomonas foetus infections in cattle

By LS

Sexually transmitted diseases in humans are something I would venture most of the adult population have experience with- if not through personal experience but through some rudimentary biology lecture on the various microbes that are prone to infecting humans following sexual contact. What may be less familiar to most people, however, are sexually transmitted diseases in cattle. One such organism that infects cows is Tritrichomonas foetus, a parasitic protozoa and the causative agent of bovine trichomonosis. Infection is characterized by spontaneous abortion and infertility. The parasite therefore represents a major threat to economic concerns of the cattle industries in the US and in Latin America and to the health of cows everywhere.
Tritrichomonas foetus is an extracellular parasite with a simple life cycle that consists mostly of a trophozoite stage (1). Trophozoites are tear drop shaped cells with a single membrane bound nucleus (2). They are able to move via the four flagella that extend from a complex on the organism’s anterior end. Three of these flagella extend freely from the anterior while the fourth extends backwards towards the posterior end (2). Trophozoites do not have a mitochondria (3) and survive best in low oxygen environments.
Under stressful conditions, Tritrichomonas foetus adopts a spherical form and internalizes its flagella (3). This psuedocyst form lacks a true cyst wall (3) and because it lacks flagella, cannot move (1). Though the role of the psuedocyst in the parasitic lifestyle is not well understood, it is thought that it exists to protect individuals from unfavorable environmental conditions, as encystment can be induced by environmental  stressors such as extreme cooling (3). However, psuedocysts are able to adhere to host cell surfaces better than trophozoites are and their adhesion is uninterrupted by treatment with antimicrobial drugs (1). It has therefore been proposed that psuedocysts may have a more active role in the Tritrichomonas foetus lifecycle than previously thought.
Infection by the parasite is localized to tissues of the reproductive system. Bulls act as asymptomatic carriers of the disease and do not experience any adverse effects or lowered fertility for being infected. Trophozoites do not invade the epithelial tissues of bulls but are present in discharge from the lining epithelial tissues of the penis, foreskin and urethra (2) and contaminate sperm. Interestingly, the age at which a bull becomes infected is a major determinant for whether or not the bull becomes a transient or chronic carrier. Experimental infection of previously uninfected bulls found that chronic infection was established frequently in bulls aged 3-7 and almost never following experimental infection of bulls aged 1-2 (2).This is not thought to be due to differences in health between older and younger bulls but is instead due to anatomical features: as bulls age epithelial crypts (indentations lined by epithelial cells) on the penis and prepuce deepen (2), creating a more favorable, lower oxygen environment for the microbe. While infection in bulls shows no clinical symptoms, it is still important because of the ability of an infected bull to spread the infection to females. Some experiments have shown that excreted products produced by T. foetus decrease the sperm motility (4). However, sperm viability is not affected(4).
Female cows acquire T. foetus infections through mating with an infected male. Trophozoites attach to epithelial cells lining the vaginal canal, uterus, and oviduct. Parasite attachment to host cells is mediated by four main surface proteins (1) though not much work has been done to characterize these proteins beyond their initial identification. Additionally, T. foetus produces soluble cytotoxins that may also play a role in establishing initial infection: a purified cysteine protease from T. foetus as well as live parasite were able to cause apoptosis in cultures of bovine vaginal epithelial cells (BVECs) (5). In cows, infection with T. foetus can interfere with egg fertilization and embryonic development (2). Spontaneous abortions in affected cows usually occur between 50 and 70 days gestation. Exact mechanisms through which this occurs have not been determined but it is likely related to the parasite’s cytotoxicity towards host cells and subsequent tissue damage and inflammation. Tritrichomonas foetus is able to cross the placenta and infect the developing embryo (6). In fact, the organism got its name because it was first identified in studies of aborted fetal calves (2). T. foetus is also able to colonize the deeper reproductive tract (such as the oviduct), resulting in tissue damage and long term infertility (6). Aside from tissue damage and infertility, overt clinical symptoms are rare. Infections are usually transient though the length of their duration can vary widely (2). Exposure does not confer long term immunity (2). In a small proportion of cases, the initial infection becomes chronic. These individuals act as reservoirs of infection and create challenges when attempting to prevent future outbreaks within herds.
Given that overt clinical symptoms are subtle in cows or non-existent in bulls, diagnosing an infection with T. foetus is difficult. It is usually done when the parasite, present in preputial or vaginal secretions, or occasionally amniotic fluid, is identified morphologically under a microscope upon direct examination of these fluids. There is no treatment for T. foetus and control methods center on preventing spread of the infection. Infected bulls are often slaughtered. Additionally, herds may be divided into infected and uninfected groups, with infected individuals being prevented from mingling with the rest of the herd. Artificial insemination has shown promise as another way to prevent the disease, as it is spread only through sexual contact. However, controlling further outbreaks after members of a herd are infected are difficult and costly. Tritrichomonas foetus therefore remains a threat to the economic health of the cattle industry, in addition to threatening the reproductive health of cows everywhere.
  

1. R. M. Mariante, L. C. Lopes, M. Benchimol, Tritrichomonas foetus pseudocysts adhere to vaginal epithelial cells in a contact-dependent manner., Parasitol. Res. 92, 303–12 (2004).
2. D. O. Rae, J. E. Crews, Tritrichomonas foetus., Vet. Clin. North Am. Food Anim. Pract. 22, 595–611 (2006).
3. A. Pereira-Neves, L. F. Nascimento, M. Benchimol, Cytotoxic effects exerted by Tritrichomonas foetus pseudocysts., Protist 163, 529–43 (2012).
4. C. M. Ribeiro et al., Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm., Theriogenology 73, 64–70 (2010).
5. B. N. Singh et al., Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells., Infect. Immun. 72, 4151–8 (2004).
6. M. Benchimol, A. B. Dias, R. Fontes, Interaction of Tritrichomonas foetus and the bovine oviduct in an organ culture model., Vet. Parasitol. 140, 244–50 (2006).