World Problem – Zoonotic Intestinal Parasites

Introduction:

Zoonotic intestinal parasites are prevalent worldwide, and one of the most common and studied is transmitted by the microscopic protozoan, Giardia intestinalis, also known as G. lamblia and G. duodenalis (Ivanov, 2010). This eukaryotic parasite is responsible for the transmission of the most common waterborne disease, giardiasis, which causes watery diarrhea, vomiting, and has even been linked to irritable bowel syndrome (Ankarklev et al., 2010). Since its discovery in 1681, Giardia has been the focus of various research in humans and animals, focusing on its unique yet simple characteristics, life cycle, and transmission pathways (Ali and Hill, 2003). This research has since identified tests and treatments for giardiasis (Rishniw et al., 2010). Giardia is an important organism for studying the various pathogens that surround us and giving insights into new molecular mechanisms (Ankarklev et al., 2010).

History:

In 1681, Antoine Van Leeuwenhoek first discovered Giardia when examining his stool sample under the newly discovered microscope (Thompson, 2008). It was not until 1859 when Lambl was able to describe the organism’s morphological characteristics for which we identify as Giardia today (Ivanov, 2010). The name Giardia was first brought to light in 1882 by Kunstler for a flagellate found in tadpole intestines. Later in 1888, Blanchard suggested that the parasite should be named Lamblia, after Lambl who described it. The names were later combined to form the genus Giardia and species Lamblia (Thompson and Morris, 2011).

The genus Giardia belongs to the order Diplomonadida and family Hexamitidae (Thompson and Morris, 2011), which are binucleated flagellates found in anaerobic or microaerophilic environments (Ankarklev et al., 2010). Within this genus are five species characterized based on host specificity and morphology: G. duodenalis, G. agilis, G. muris, G. ardeae, and G. psittaci. The only species capable of parasitizing humans is G. duodenalis (Ivanov, 2010; Thompson, 2008). Based on allozyme electrophoresis, scientists have been able to genotype Giardia, and further differentiation of these genotypes has been established by polymerase chain reaction techniques (Thompson, 2008). G. duodenalis was determined to contain eight genotypes that are similar in morphology but differ in host specificity: A, B, C, D, E, F, G, and one group that is unnamed. Within these genotypes are subgroups, for example, genotype A contains two subgroups, AⅠ and AII. AI is comprised of humans and animals that are closely related and has high zoonotic potential while BⅣ is human-specific. There have been cases of a mix of subgroups B and C where close inhabitants, canines and humans, were infected (Ivanov, 2010).

Eukaryotic organisms are complex and must contain a nucleus and membrane-bound organelles. With two nuclei, an endomembrane system, and a complex cytoskeleton, Giardia meets these requirements. However, by lacking typical higher eukaryotic organelles – mitochondria, peroxisomes, and the Golgi apparatus (Lujan and Touz, 2003), some refer to Giardia as the “link” between prokaryotes and eukaryotes (Ankarklev et al., 2010). The organism’s parasitic lifestyle is thought to be the cause of the lack of complex organelles (Lujan and Touz, 2003). The simplicity of this parasite is an important characteristic for researchers studying eukaryotic organisms as it allows the cell cycle to be studied outside of the host (Ankarklev et al., 2010).

Characteristics: Giardia can be observed in two life forms: trophozoite and cyst. The trophozoite, which is motile and never seen outside of the host, is characterized by two diploid nuclei, four pairs of flagella, and a unique organelle called the adhesive disc (Thompson and Morris, 2011). It is known for its teardrop shape and is approximately 15 μm long (Ali and Hill, 2003). The cyst, the infectious form, is non-motile, oval-shaped, contains four nuclei (Ivanov, 2010), and is protected from harsh conditions by a hard cell wall (Thompson, 2008).

The adhesive disc, flagella, and the median body compose the cytoskeletal component of Giardia. The cytoskeletal elements, microtubules, and microfilaments allow the parasite to move through the host’s digestive tract and attach to the host’s epithelial cells for reproduction and pathogenesis. They also aid in intracellular transport, cell division, and morphological changes throughout the life cycle (Dawson, 2010). The microfilaments within the cytoskeleton lack actin-binding proteins for regulation that are normally found in eukaryotic organisms (Paradez et al., 2011), while the microtubules contain giardins, a special family of proteins that are specific to Giardia and localize around the adhesive disc (Dawson, 2010).

The adhesive disc is critical to the virulence of the parasite (Dawson, 2010) and is important for cell attachment to the host’s intestinal epithelial cells (Ankarklev et al., 2010). It is a highly organized microtubule structure, composed of three elements: a spiral array of microtubules, microribbons, and cross-bridges, all of which are thought to undergo conformational changes that aid in the attachment of the parasite to the microvilli of the host (Dawson, 2010). The median body is unique to Giardia and is thought to serve as a tubulin reservoir for allowing quick formation of adhesive discs during replication, but no experiments have been performed (Dawson, 2010). Finally, the eight flagella are organized into pairs on the anterior, caudal, posteriolateral, and ventral sides. Giardia flagella are composed of the usual axoneme structure. However, each pair of flagella has its specific proteins and structures associated with them, and the basal bodies at the base of the flagella are all centered between the two nuclei (Sagolia, 2006; Dawson, 2010). Flagella make it possible for the cell to migrate, making it a virulence factor, and also aid in cell division (Dawson, 2010).

Life Cycle

Transmission of Giardia occurs most commonly through ingestion of contaminated water. However, with poor hygiene, it can be passed through consumption of fecal material (Ivanov, 2010; Leibly et al., 2011). Animals are considered reservoirs of the pathogen, but human-to-human transmission is the most significant pathway in high-frequency areas (Thompson, 2008). The most commonly seen cases in humans are in developing countries where water treatment procedures are inadequate. But it is also seen in children, who have the highest risk of infection (Leibly et al., 2011). Giardia is often seen in areas with damage or flaws to the filtration systems and where water for irrigation is used for raw food processing (Ivanov, 2010). Giardia is also commonly seen in domestic animals, usually canines in shelters or breeding facilities (Rishniw et al., 2010).

The life cycle contains two stages: excystation and encystation, where the cell must duplicate and divide out both of the diploid nuclei and multiple cytoskeletal features (Sagolia et al., 2006). Prior to excystation, the cyst stage is outside of the host, encapsulated by a protective membrane, where it can survive in cold water for long periods of time. This “time-released capsule strategy” is characterized at first by a dormant cyst with down-regulated metabolism and low gene expression (Faghiri and Widmer, 2011). Once the cyst is ingested by the host, it passes through the stomach, and while still intact, stomach acids trigger the cyst to become metabolically active and excystation begins (Ankarklev et al., 2010). It is thought to be initiated by protein kinase A (Ali and Hill, 2003). The cyst passes into the small intestines, and cysteine proteases released within the cyst wall allow the cyst to rupture near the poles first, then the excyzoite body is released (Ankarklev et al., 2010).

The excite is now able to undergo two consecutive cycles of cytokinesis without DNA replication, while simultaneously increasing metabolism and gene expression. Excystation ends with four binucleated trophozoites, each containing the adhesive disc that binds to the intestines of the host (Ankarklev et al., 2010). The attachment of the trophozoite to the microvilli is important for the cell to proliferate and avoid being forced into the colon by peristalsis (Dawson, 2010). The mechanism for this is unknown, but is thought to be the result of negative pressure underneath the ventral disc, which is caused by conformational changes in the disc structure (Dawson, 2010). While the parasite is attached to the host, the symptoms of giardiasis will occur until the trophozoite releases. The trophozoite can survive until it can no longer attach to the microvilli and enters the lower part of the small intestine as encystation begins and the cell begins to transform back into the infective cyst stage (Ankarklev et al., 2010).

Encystation of the trophozoite is necessary for the survival of Giardia outside of the host (Bittencourt-Silvestre et al., 2010). For encystation to begin, there must be a stimulus such as the deprivation of cholesterol and bile at a high pH. When mature intestinal epithelial cells slough off, trophozoites are released and exposed to increased bile and a higher pH than when attached (Lauwaet et al., 2007). The flagella become internalized, and the adhesive disc loses its ability to attach to the host (Ankarklev et al., 2010), which is caused by the disassembly of the microtubule spiral within the structure (Dawson, 2010). The trophozoite becomes encysted as dipeptidyl peptidase IV-associated proteolysis and signal transduction induces the expression of encystation-specific genes, which help regulate the expression of secreted cyst wall proteins (Ali and Hill, 2003). These cyst wall proteins are synthesized and exported to the exterior of the cell by encystation-specific vesicles (Lauwaet et al., 2007).

These vesicles are thought to be similar to the Golgi apparatus, an organelle that is involved in protein trafficking (Ankarklev et al., 2010). The cyst wall proteins undergo post-translational modifications by the use of five protein disulfide isomerases, lysosomal cysteine proteinases, and a calcium-binding granule-specific protein (Lauwaet et al., 2007). There are four known cyst wall proteins; three contain leucine-rich repeats with cysteine residues, and the fourth resembles trophozoite variant surface proteins (Reiner, 2008). The cell wall forms as the cell undergoes DNA replication to form two tetraploid nuclei. These nuclei then divide without cytokinesis (Ali and Hill, 2003) to give four diploid nuclei, which are replicated again to form four tetraploid nuclei per cyst. This is regulated by cyclin-dependent kinase-1 and Cyclin B (Ali and Hill, 2003). Each cyst is now in the dormant and infectious stage, with low metabolic activity and low gene expression. The cysts are then released into the environment when excreted with the host’s fecal matter, where they wait to be ingested by another host to begin another life cycle (Ivanov, 2010; Thompson, 2008).

When contracting Giardia, symptoms of the pathogen will result from the proliferation of the parasite in the intestines. Once the trophozoite attaches to the host’s intestinal epithelial cells, it replicates by binary fusion and colonizes in the intestines. Within nine to fifteen days, giardiasis will begin. Many theories exist for the mechanism that causes giardiasis. Ivanov (2010) suggests that the metabolites released by the trophozoite destroy important peripheral membrane proteins, which damages the brush border. In another study, Chin et al. (2002) suggested the parasite disrupts the zona-occludens of tight junctions in the epithelial cells, but Scott et al. (2002) determined that giardiasis was caused by myosin light chain kinase-dependent phosphorylation of F-actin and zona-occludens. All of these theories result in increased epithelial permeability, which ultimately causes the gastrointestinal problems associated with giardiasis (Ivanov, 2010; Chin et al., 2002; Scott et al., 2002). The severity of giardiasis varies depending on which Giardia subgroup is responsible and the host’s immune response. For example, young animals with deficient nutrition are more likely to suffer mortality when compared to mature healthy animals (Ivanov, 2010).

The clinical symptoms of giardiasis can vary from asymptomatic, acute to chronic infection (Ali and Hill, 2003). It is usually associated with acute to chronic diarrhea with weight loss, dehydration, and abdominal pain. The acute stage will begin with a low-grade fever and nausea and last three to four days. The parasite will also cause severe diarrhea, resulting in dehydration and severe abdominal cramping. If left untreated, these symptoms can persist for months. The chronic stage is more severe and is characterized by headache, muscle pain, progressive weight loss, and malabsorption. These two stages are variable, usually unpredictable, and sometimes intermittent (Ivanov, 2010). Symptoms can pass on their own or when interrupted by conventional medications that disrupt the cell’s life cycle.

Diagnosis, Treatments, and Prevention

When apparent symptoms of Giardia are present, there are many ways to diagnose this pathogen, as well as multiple treatments for both humans and animals. Typically, diagnosis involves the presence of the cyst shedding from the host’s intestinal tract (Ivanov, 2010; Rishniw et. al., 2010). Fecal flotation in ZnSO4 and centrifugation are typically used for the detection of this pathogen; however, cyst shedding is intermittent. Therefore, fecal samples can be unreliable unless multiple fecal samples are tested. A more reliable diagnostic tool is antigen assays (Rishniw et. al., 2010). Direct immunofluorescent coproscopy, also known as direct fluorescence antibody (DFA), uses fluorescent antibodies to detect Giardia (Rishniw et. al., 2010; Ali and Hill, 2003). Another antigen assay, enzyme immunoassays (EIAs), detects soluble stool antigens (Ali and Hill, 2003). Other antigen tests are also used to detect the excreted copro-antigen. Such tests are found in veterinary medicine as Quantitive ELISA and IDEXX SNAP tests. Antigen diagnosis is more sensitive and specific when compared to fecal examinations (Rishniw et. al., 2010).

When diagnosed with Giardia infection, common treatments such as nitroimidazoles and benzimidazoles are commonly used for human infections, while canine and feline infections are commonly treated with benzimidazoles (Thompson, 2008). Ultrastructural affects on the trophozoite life form are induced, such as distorted shape, microtubule and microribbon distortion, and swelling of the trophozoite, affecting the attaching abilities of the adhesive disc and eventual fracture of this crucial structure (Ali and Hill, 2003). Alternative treatments such as probiotic therapy have also been useful by inhibiting proliferation of the trophozoites (Thompson, 2008). Resistance to the current treatments has caused much concern, resulting in new treatment therapies being pursued (Leibly et. al., 2011).

Recent research into new treatments for Giardia has discovered the importance of kinases as new drug targets. Cyclin-dependent kinases are associated with the cell cycle, known for their involvement in regulation, and have been the focus of many cancer treatments. Researchers have discovered that the inhibition of cyclin-dependent kinases leads to cell apoptosis. Giardia contains its own cyclin-dependent kinases; therefore, researchers are currently investigating this as a new drug target for the treatment of Giardia infection (Leibly et al., 2011).

Prevention mechanisms are of great importance in developing countries to prevent or end the epidemic of Giardia transmission. Once treated with medications, the cysts are shed into the environment and can become infectious once ingested. Prevention of this continued infectious stage may be possible through the regulation of gene expression. Using antigenic variation, a mechanism that Giardia uses to turn genes on or off to evade the host’s immune response, researchers have been able to find a potential target for a vaccine against the parasite. Variant-specific surface proteins are located on the surface of the trophozoite (Ankarklov et al., 2010), and with histone modifications such as hyperacetylation or deacetylation, chromatin structures are altered and can result in transcriptional silencing of genes on these proteins.

Researchers have studied this to determine if the altered gene expression of these proteins can result in the inhibition of cyst formation, the infectious stage of Giardia. They discovered that the encystation-specific proteins contained short-flanking regions, which were important target areas for drugs to alter gene expression. They found that when Giardia’s histone modification abilities were inhibited, encystation was blocked, and the parasite was unable to complete its life cycle of formation into a cyst. This epigenetic regulation can be a useful tool for pharmaceutical companies to produce a therapy that will prevent this parasite from causing future epidemics (Sonda et al., 2010; Rivero et al., 2010).

Conclusion

Giardia is a highly reduced, somewhat simple protist that is the cause of one of the most common parasitic infections, giardiasis. This water-borne pathogen contains several genotypes and may have zoonotic potential and causes multiple gastrointestinal problems while proliferating inside of the host. The simplistic nature of this organism has brought much attention to its usefulness in studying eukaryotic organisms. With a two-stage life cycle, excystation results in the release of the mobile trophozoite into the small intestines of the host, which eventually becomes encysted into the infectious cyst form as it leaves the host. This continuous cycle has the ability to cause epidemics in developing countries and in some animal populations, leaving many researchers to search for better therapies and possible vaccinations. Giardia’s characteristics and simplicity give insight into the potential for future discoveries in more complex higher eukaryotic organisms.

Literature Cited

1. Ali, S.A., D.R., Hill. 2003 Giardia intestinalis. Current opinion in infectious diseases 16: 453-460.
2. Ankarklev, J., Jeristrom-Hultqvist, J., Ringqvist, E., Troell, K., and S.G.
   Svard. 2010. Behind the smile: cell biology and disease mechanisms of Giardia species. Nature Reviews: microbiology 8: 413-422.
3. Bittencourt-Silvestre, J., Lemgruber, L., W. de Souza. 2010. Encystation process of Giardia lamblia: morphological and regulatory aspect. Archives of microbiology 192: 259-265.
4. Chin, A.C., Teoh, D.A., Scott, K.G., Meddings, J.B., Macnaughton, W.K., A.G. Buret. 2002. Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase- 3-dependent manner. Infection and immunity 70: 3673–3680.
5. Dawson, S.C. 2010. An insider’s guide to the microtubule cytoskeleton of Giardia. Cellular microbiology 12: 588-598.
6. Faghiri, Z. and G. Widmer. 2011. A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays. BMC Microbiology 11: 91-99.
7. Ivanov, A.I. 2010. Giardia and giardiasis. Bulgarian journal of veterinary medicine 13: 65-80.
8. Lauwaet, T., Davids, B.J., Reiner, D.S., F.D. Gillin. 2007. Encystation of Giardia lamblia: a model for other parasites. Current opinion in microbiology 10: 554-559.
9. Leibly, D.J., Newling, P.A., Abendroth, J., Guo, W., Kelley, A., Stewart, A.J., W. Van Voorhis. 2011. Structure of a cyclin-dependent kinase from Giardia lamblia. Structural biology and crystallization communications 1: F1-6.
10. Lujan, H. D., M.C. Touz. 2003. Protein trafficking in Giardia lamblia. Cellular microbiology 5: 427-434.
11. Paredez, A. R., Assaf, Z.J., Sept, D., Timofejeva, L., Dawson, S., Wang,
    C.R., W.Z. Cande. 2011. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins. PNAS Early Edition: 1-6.
12. Reiner, D. S. 2008. Cell cycle and differentiation in Giardia lamblia. Thesis- Department of microbiology, tumor and cell biology, Karolinska Institutet, Stockholm, Sweden.
13. Rishniw, M., Liotta, J., Bellosa, M., Bowman, D., K.W. Simpson. 2010. Comparison of 4 diagnostic tests in diagnosis of naturally acquired canine chronic subclinical giardiasis. Journal of veterinary internal medicine 24: 293-297.
14. Rivero, F.D., Saura, A., Prucca, C.G., Carranza, P.G., Torri, A., H.D. Lujan. 2010. Disruption of antigenic variation is crucial for effective parasite vaccine. Nature medicine 16: 551-558.
15. Sagolla, M., Dawson, S., Mancuso, J., W. Cande. 2006.Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. Journal Of Cell Science 119: 4889-4900.
16. Scott, K.G., Meddings, J.B., Kirk, D.R., Lees-Miller, S.P., A.G.Buret. 2002. Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterol 123: 1179–1190.
17. Sonda, S., Morf, L., Bottova, I., Baetschmann, H., Rehrauer, H., Caflisch, A., Hakimi, M.A., A.B. Hehl. 2010. Epigenetic mechanisms regulate stage differentiation in the minimized protozoan Giardia lamblia. Molecular microbiology 76: 48-67.
18. Thompson, R.C.A. 2008. Giardiasis: modern concepts in control and management. Annales nestle 66: 23-29.
19. Thompson, R.C.A. and P.T. Morris. 2011. Taxonomy of Giardia species.
    Giardia: a model organism: 3-12.