- 1 Classification
- 2 Description and Significance
- 3 The Organism
- 4 Pathology
- 5 Current Research for Diagnosis and Treatment
- 6 References
Higher order taxa
Eukaryota; Stramenopiles; Oomycetes; Pythiales; Pythiaceae; Pythium
Description and Significance
Pythium insidiosum is a pathogenic species of water mold (oomycete) that infects humans and animals, primarily horses, cattle, dogs, and cats . This microorganism is usually found in wet, swampy areas with dense vegetation . Exposure to this organism occurs via contact with the water in which it lives as well as plants it is living on. Infection by this pathogen leads to a disease called Pythiosis, which is characterized by large lesions on the skin and in tissues such as the gastrointestinal tract; it also has a high mortality rate . The issue of high mortality makes this pathogen a public health concern because infection advances quickly once it occurs; thus, early detection is crucial. P. insidiosum is well adapted to invading and thriving in its mammalian and plant host’s tissues; its life cycle and motile zoospores increase its pathogenicity as well as the rate at which infection progresses. To address this issue, substantial research has been conducted on early detection methods using polymerase chain reaction, antigen identification, and other molecular and serological assay techniques   . Scientists have information about the organism itself in terms of its morphology and pathogenicity, yet more information is needed for early detection of this organism and rapid identification in a host, as well as genetic and structural information regarding this organism’s genome. P. insidiosum currently kills large amounts of livestock and food crops in tropical developing nations, leading to high rates of infection and death in humans living in countries such as Thailand where agricultural land is swamp-like  , and affects domesticated pets in southern parts of the United States . These events are presenting both economic and public health concerns that call for immediate attention.
The genome of P. insidiosum has not been sequenced . Genomes of oomycete clades vary widely in size, some containing 19 to 21 million base pairs while others contain between 220-280 million base pairs, many of which make up non-coding DNA . However, genomic sequencing of other species in the genus Pythium (to which this organism belongs) has revealed that some organisms in this genus have genomes that contain around 43 million base pairs with about 15,000 protein-coding genes with only 7 percent of the genome being repetitive DNA sequences . As an oomycete, P. insidiosum is eukaryotic and its DNA is packed into chromosomes in a membrane-bound nucleus. Additionally, this organism is capable of carrying out horizontal gene transfer with some fungal species .
This organism forms hyphae, which is a common structure of fungi; however this organism is not a true fungus for several reasons . First, the cell wall does not contain chitin, but instead is composed of cellulose and β-glucans . Second, the plasma membrane of the cells lacks ergosterol, which is a compound commonly found in fungi that helps convert alcohols into a usable form of vitamin D2 under ultraviolet light . Third, this organism’s cells are diploid in its vegetative state, while fungi are usually haploid in their vegetative state . In fact, this organism, as an oomycete, is more closely related to kelp, specifically brown algae of the phylum Phaeophyta . As for cell morphology and components, the cells of this organism are eukaryotic and have a round and spherical shape that includes many filamentous projections, vacuoles, a cell wall, and membrane bound organelles such as mitochondria, endoplasmic reticulum, and others like those found in other eukaryotes . Broad hyphae that are arranged at right angles projecting off of the main cell body are a distinguishing morphological characteristic of this organism .
Life Cycle and Zoosporogenesis
Pythium insidiosum is capable of reproducing asexually in plant and animal tissues as well as in aquatic environments, by producing sporangia and zoospores . These structures are produced quickly in warm, moist conditions. Research indicates that it only takes about 35 minutes for the zoospores to be released once the sporangia are produced. During its asexual cycle, P. insidiosum creates sporangia at the end of its hyphae, and the protoplasm containing cytoplasmic fluids, ions, and organelles flows to the hyphal tip; the vesicle at the tip increases in size until the protoplasm stops flowing . At this point, the base of the vesicle where it connects to the hyphal tip forms a cleavage and then forms biflagellate zoospores inside the vesicle . The motile zoospores mechanically break the vesicle’s wall and are released into the surrounding environment . They are globose to ovoid in shape, have two flagella, and swim around from anywhere between 10-60 minutes before encystment, depending on the environment  . They exhibit chemotaxis toward a favorable nutrient or substrate in an environment, and show more significant chemotaxis toward mammalian and plant tissue as well as animal hair; it is even more pronounced when the tissue is damaged . Over time, zoospores slow down and settle in a particular place, where their two flagella detach, they encyst on a surface such as a piece of tissue, adhere to the surface by secreting a glycoprotein, and develop a germ tube that elongates into a filament like those of the original mature organism . In environments containing plant and animal tissues, the zoospores move more directly and encyst more quickly, most often directly onto the tissues. Movement is much more random and encystment and germ tube production occur much more slowly in environments that only contain a control substance like water without nutrients or useful substrates . This phenomenon shows how preferred these tissues are for this organism, which gives evidence about its pathogenicity and its ability to infect larger hosts quickly as a result of this strong exhibited preference.
P. insidiosum uses its filamentous hyphae for feeding and nutrient uptake from the surrounding environment . Because it lives in swampy ecosystems, it often decomposes decaying organic matter for nutrients. P. insidiosum also conducts aerobic respiration . During decomposition, it secretes different enzymes and compounds into the surrounding environment and uses water and its hyphae to get nutrients to flow back to the organism to be digested . However, the specific details of this mechanism are not yet described or understood. Metabolic activity is stimulated by the presence of magnesium, potassium, and calcium ions . Additionally, P. insidiosum has a high affinity for iron, which can expedite or stimulate metabolic processes . This activity is especially useful when P. insidiosum invades a host and depletes the resources available there, because iron can be readily available in host physiological environments .
P. insidiosum has a global distribution and is found in the tropics, subtropics, and some temperate regions . It is present in ecosystems in Southeast Asia, South and Central America, Africa, and North America . Its distribution is expanding because the climate in many areas of the world is becoming more tropical and warmer as a result of climate change. P. insidiosum is predominantly found in tropical wetland environments as well as similar wet, densely vegetated, swamp-like areas where a lot of decaying organic matter from plants is available to break down for nutrients . It is also commonly found in soil, on decaying plant and animal materials, and in aquatic ecosystems such as ponds as well as grasses around the edges of them . The aquatic environment is ideal for the production of zoospores, which can swim though the water, encyst on a surface, and germinate hyphae and start a new colony. P. insidiosum is well adapted to living in temperatures between 34ºC and 36ºC as well as 40ºC and 45ºC, and forms transparent, faintly white colonies in favorable environments . Its primary ecological role is a decomposer (and thus a heterotroph), gaining most of its energy by decomposing decaying plant and animal matter and extracting organic sugars and other compounds for energy . It is also a plant pathogen, infecting damaged plant tissues. Animals are susceptible to infection as well if they come into contact with P. insidiosum colonies in water or on plants.
P. insidiosum zoospores are the main vehicle for infection of host tissues  . They exhibit strong chemotaxis to damaged tissues including hair, skin, plant tissue, and the gastrointestinal tract . Infection occurs by the host being in direct contact with water or plants containing zoospores. Ingestion of water containing zoospores also leads to infection. Susceptibility is increased if the host already has a lesion or damage on the surface of the skin. Small puncture lesions from insect bites also provide an entry point for P. insidiosum  . Some studies have shown that this oomycete is able to infect insects, which then transmit its cells and zoospores into the host bloodstream when they bite a host organism . This presents an epidemiological concern with mosquitoes because both the organism and the mosquito are prevalent in wet, tropical environments.
Zoospores infect the host skin by swimming to it and encysting on the surface of [damaged] tissue . They adhere to the surface and are stimulated by the host’s warm body temperature to produce a germ tube (hypha) that extends into the tissue and eventually into local blood vessels, allowing for infection of additional tissues around the body . The cells quickly mature and expand hyphae into surrounding vessels and tissues for increased nutrient uptake and rapid tissue colonization; it is this rapid spread throughout the host’s body that makes this organism such a significant public health concern. Invasion of the vascular system leads to thrombosis and invasion of the large arteries . Its virulence is increased by the secretion of proteases and the mechanical force that the hyphae place on the host’s body tissues . Studies show that the proteases released act on the tissue to create a dramatic decrease in tissue strength, allowing for hyphae to easily expand into host body tissues . This damages the host’s tissues and allows the organism to colonize larger areas in the body, depleting structure and function of tissues and killing them. Ultimately, cells continue to release zoospores which form more cells, and cells continue to grow and multiply. This leads to local inflammation in response to the invading cells and a cancer-like mass develops (leading to this disease’s more common name, “swamp cancer”) . The mass continues to grow and spread to surrounding tissues, killing them and killing the host when vital tissues (such as heart, brain, and others) are killed . Inside the host, the three P. insidiosum serine proteases differ in molecular weight and specificity . This allows for the proteases to attack different parts of the host’s body as well as different types of tissue at the same time . Thus, infection by P. insidiosum and the development of Pythiosis is very rapid, making early detection very important . Without early detection, the risk of death is very high with Pythiosis. Progression of the infection and its severity occurs very quickly, so detecting the organism as close to the time of onset of infection as possible is crucial .
Issues with Current Treatment
Current treatment for Pythiosis includes amputation of the infected limb, excision of infected tissue or the lesion, prescription of antifungal medications, immunotherapy, or a combination of these . Excision temporarily relieves the host of the condition, yet P. insidiosum often returns some time later, showing that it is difficult to free the body of P. insidiosum after infection has occurred. Anti-fungal medications only slow infection without eliminating the organism . Immunotherapy has shown to cause inflammation without destroying the organism because it is able to create antigens and other toxins that lead to immune responses but eventually compromise the immune system, which can lead to other diseases . It is not a true fungus so anti-fungal medications are not a very effective, permanent treatment. The ineffectiveness of treatment is a result of the rapid progression of the disease.
Current Research for Diagnosis and Treatment
Much of today’s research on this organism is focused on early detection and identification of P. insidiosum within the host so that the condition can be treated before it gets to a stage of infection where remedies and therapies are no longer useful. Identification of the organism in the host can be difficult and time consuming, so research is focusing on faster detective methods.
Some research is focused on the development of primers for a PCR assay that can be used to amplify the organism’s DNA and RNA and sequence them for fast identification . Issues with this development are that the assay can sometimes give false positive results, so research is continuing to make the assay more specific for identification via a two-step process, the first to identify fungal DNA and the second to specifically identify the DNA and presence of P. insidiosum . Other research is dedicated to detecting a specific antigen of P. insidiosum in the host’s body using Western blotting and electrophoresis . Scientists have already recognized a 74-kilodalton antigen that is present in all P. insidiosum isolates, so more research is being developed to use this antigen for detection . Other research is dedicated to developing specific staining for this microorganism and improvement on morphological identification by comparing P. insidiosum isolate morphology and staining in tissues across different mammalian and plant hosts so that a simple and fast detection method can be developed with staining and observation . Lastly, further research is being conducted to quickly identify P. insidiosum isolates with 18S RNA sequencing and other Western blotting techniques to isolate P. insidiosum, its antigens, and genetic components .
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Created by [Lindsay Smith], student of Jennifer Talbot for BI 311 General Microbiology, 2014, Boston University.