Protozoa- Protista

Over 45,000 species described- many parasitic

Parasitic protozoa kill, mutilate, and debilitate more than any other group of disease organisms

Protozoa used to be a phylum so many references will use protozoa and protista interchangeably

Protozoa

Single cells

Some have >1 nucleus during all or part of their life cycles

Some stages (spores) may be constructed from >1 cell

Largely recognized after the development of microscopes

van Leeuwenhoek described all sorts of protozoa

Electron microscopy indicated that all protozoa did not have a common body plan- used for grouping organisms into different phyla

Protozoa possess a great diversity of membranous organelles in the cytoplasm

Mitochondria- organelles with enzymes of the oxidative phosphorylation system and tricarboxylic acid cycle-diversity in shape of mitochondria

Golgi apparatus-elaborate and diverse in different protozoa-may occur as single or multiple large parabasal bodies often in association with kinetosomes. Golgi can play diverse roles in protozoa: protein assembly, source of skeletal plates and polar filaments

Membrane-bound bodies:

Microbodies- spherical structures with a dense granular matrix- often contain oxidases and catalases

Bodies often named due to role:

Peroxisomes: Oxidase: reduces oxygen to H2O2

Catalase: converts H2O2 to water and oxygen

Hydrogenosomes produce molecular hydrogen

Microbodies may contain enzymes for glyoxylate cycle fat-carbohydrate conversion

Reproduction

Asexual or sexual

Asexual: binary fission: plane may be random (Sarcodina) longitudinal in flagellates (between the kinetosomes or flagellar rows)

Nuclear division is mitiotic.

Multiple fission (merogony, schizogony)-nucleus and other essential organelles divide repeatedly before cytokinesis- producing simultaneously a large number of daughter cells.

In shizogony-the cell is called a shizont and daughter nuclei arrange themselves peripherally beneath the cell surface. Daughter cells are called merozoites. Merozoites are additional stages of the same life-cycle stage.

Sexual Reproduction

Reductional division in meiosis: diploidy-haploidy

gametes join to restore diploidy

Amphimictic: union of gametes from 2 parents

Automictic: one parent produces both gametes.

Many protozoa protected within cysts- resistant stage covered in chitin or chitin-cellulose mixes.

Triggered by: temperature, adverse environment, dessication, lack of food, pH. In some situations we do not know what is the trigger.

Entamoeba histolytica can be spread by persons who are asymptomatic, but are shedding cysts in their faeces.

Under suitable conditions- excyst

-triggered by change in environment: water, temperature, pH, host digestive enzymes, parasite produced enzymes

Feeding / Metabolism

Some protozoa are holophytic – synthesize carbohydrates.

Heterotrophic- require energy in form of complex carbon molecules, nitrogen as preformed amino acids

Most are particle feeders-grazers and predators

Mouth openings may be temporary (amoebas) or permanent cytostomes (ciliates)

Food enters food vacuole, digestive organelle that forms around ingested food – undigestible material voided through temporary opening or permanent cytopyge (ciliates)

Endocytosis: internalizing material:

Pinocytosis-liquid, phagocytosis-particulate matter

Protozoa carry out glycolysis, Krebs cycle, ETS, transaminations, nucleic acid synthesis etc.

ATP is principal form of available energy

Glycogen is common energy storage

Genes are transcribed in nucleus, peptides synthesized on ribosomes as in other systems

Many parasites live in reduced oxygen environments. Often they derive energy from glycolysis, and partially oxidized products excreted

Non Pathogenic Protozoa:

Each day all of us are infected with bacteria-viruses-protozoa- parasites

Vast majority expelled due to their inability to take advantage of our essential and available niches.

Many protozoa are commensals:

Non pathogenic, flagellated commensals of humans:

Trichomonas tenax, T. hominis, Enteromonas hominas, Retortamonas intestinalis, Chilomastix mesnili

Commensal Amoebae

Entamoeba dispar, E. hartmanii, E. coli,

Iodamoeba butschlii

Giardia lamblia

Amitochondriate flagellated protozooan

Transmitted via fecal-oral route

Aerotolerant anaerobes and require a reducing environment

First known description by Van Leeuwenhoek in 1681 in stool samples

Two forms:

Trophozoite and cyst

Trophozoite is pear shaped, 10-20 µm long, 7-10 µm diameter

8 flagella

Binucleate- both nuclei are transcriptionally active

2 rigid median bodies

No mitochondria, peroxisomes, hydrogenosomes or other subcellular organelles for energy metabolism

Anterior region contains structure for attachment to epithelial cells

Structure is maintained with tubulin and giardins (calcium binding annexins)

Surface is covered with cysteine-rich molecules

Infection: ingestion of cysts.

Cysts excyst in response to physiological / environmental stimuli

Following a series of stimuli: acid, pancreatic enzymes

Stimuli activates the parasite within the cyst (quadrinucleate)

Motile parasite divides into 2 binucleate parasites

Trophozoites attach to epithelial cells- probably based on carbohydrate-binding lectins

But will also attach to glass plates

Parasites grow-divide by binary fission.

Giardia attaches to cells in intestine

In the presence of trypsin trophozoites produce lectins

Uses lectins to bind to cells

This interaction may delineate where parasite occurs in the hosts

Nutrition

G. Lamblia cannot synthesize nucleic acids de novo.

No mitochondria, tricarboxylic acid cycle

Uses glucose and arginine as major energy sources

Obtains these from host- often through degradation of host mucous

Lipids are absorbed directly or via endocytosis

Food reserves are stored in the form of glycogen. Glucose catabolism via the glycolytic pathway results in production of the end products ethanol, acetate and carbon dioxide.

Some trophozoites encyst.

Can be induced via exposure to bile and elevated pH

Passed out in feces

Cyst production continues for years- numbers produced vary depending on many factors

Cysts are resistant to many treatments- mild chlorine

Can survive in cold water

Boiling can kill- but not at high altitudes

Pathogenesis

Steatorrhea

Malabsorption

Associated weight loss

Physiological changes experienced during symptomatic infection could relate to host-based responses

Antigenic variation of trophozoite surface components occurs- changes every 12 G

Helps parasite avoid elimination by humoral responses of the host (IgA antibodies) directed towards the trophozoite surface proteins.

Switching of cysteine-rich variant surface proteins (VSP) occurs during excystment- allowing parasite to avoid immune response

G. Lamblia in immune deficient mice often do not show VSP switching- or at least not to the extent shown in normal hosts but may occur spontaneously or in response to physiological processes

Constant interactions between host and parasite:

mother’s milk is protective

Lactoferrin/products produced by lipid hydrolyisis of milk are toxic

Nitric oxide produced by epithelial cells is toxic

Arginine required to produce nitric oxide- if Giardia can compete for arginine- less nitric oxide produced

Duration and severity of infection depends on the immune responses of the host and the parasites’ response

Highly contagious

Most infected people are asymptomatic but may become carriers

Symptoms: protracted diarrhea- semi-solid stools to pure liquid

There is no blood loss associated with the diarrhea, however, the stool characteristically has a fatty consistency (steatorrhea) as a result of fat malabsorption. This occurs in heavy infections where attached trophozoites can cover much of the intestinal epithelial surface.

Can last months

Chronic infections: malabsorption- with substantial weight loss, debility, constant fatigue

Other symptoms include epigastric discomfort, pain, increased intolerance or allergic responses to specific foods but no cause/effect relationship has been established

How do we get Giardia???

By putting something in your mouth or accidentally swallowing something that has come in contact with the stool of a person or animal infected with Giardia.  By swallowing recreational water contaminated with Giardia. Recreational water is water in swimming pools, hot tubs, jacuzzis, fountains, lakes, rivers, springs, ponds, or streams that can be contaminated with sewage or feces from humans or animals.

By eating uncooked food contaminated with Giardia. Thoroughly wash with uncontaminated water all vegetables and fruits you plan to eat raw.

By accidentally swallowing Giardia picked up from surfaces (such as toys, bathroom fixtures, changing tables, diaper pails) contaminated with stool from an infected person.

Giardiasis is most frequently associated with the consumption of contaminated water. Outbreaks have been traced to food contamination by infected or infested food handlers, and the possibility of infections from contaminated vegetables that are eaten raw cannot be excluded. Cool moist conditions favor the survival of the organism.

Giardiasis is more prevalent in children than in adults, possibly because many individuals seem to have a lasting immunity after infection. This organism is implicated in 25% of the cases of gastrointestinal disease and may be present asymptomatically. The overall prevalence of infection in the United States is estimated at 2% of the population. The disease is also common in child day care centers, especially those in which diapering is done. (EFFECT OF CROWDING)

Who is susceptible?

Everyone: Immunocompromised patients- HIV/AIDS, cancer patients, transplant rejection patients, cystic fibrosis, malnourished individuals

Diagnosis

Microscopic examination of stool

trophozoite

cysts

Treatment.

The drug of choice for the treatment of giardiasis remains Metronidazole (Flagyl), but quinacrin hydrochloride and furazolidone are also frequently used.

However, drug resistance has been observed with each of these compounds. In addition, toxicity has restricted their use in women of child-bearing age, furazolidone has been used preferentially for children as it is can be administered as a suspension. Nevertheless, this compound has been recognised by the FDA in the U.S. as both a mutagen and carcinogen and can no longer be used.

Entamoeba histolytica

Amitochondriate ameba

Causes diarrhea and dysentery

Rarely- spreads to extraintestinal sites

Acquired by ingesting contaminated feces – in food / water

Exists as a trophozoite in host and cysts outside host

Some animals can become infected - none serve as important reservoirs

Entamoeba dispar

Morphologically identical- often misidentified

Patients with diarrhea often tested- misdiagnosed and treated

Trophozoite is motile

Facultative anaerobe

Uses glucose as energy source

Uses anaerobic metabolic pathways

Trophozoites have single nucleus and lysozomes

Cysts are smaller, no more than 4 characteristic nuclei

Described in 1800’s

Schaudinn 1903 depicted trophozoites and cysts and promptly died from overwhelming amebiasis

Direct Life Cycle

Cysts ingested- one is enough to begin an infection (one of the most efficient protozoa)

Excyst in small intestine- why?

Receives environmental cues- acid/basic

Stimuli required to excyst- release 4 trophozoites.

Carried to large intestine

Penetrate perimucosal space- attach to epithelial cells via lectin carbohydrate interactions- cytotoxic.

They engulf and lyse living cells- causing ulcers- can result in hematogenous or lymphatic spread

Some trophozoites encyst inside lumen- pass out in feces

Cysts can survive weeks in warm moist conditions

E. Histolytica

Adheres to colon cells

Secretes enzymes-kills/damages cells

Phagocytosis of cellular debris

Entamoeba histolytica must attach to host tissues

Attachment based on interactions between epithelial cell membrane-bound

N-acetyl-glucosamine and N-acetyl-galactosamine and two surface lectins

The genes for parasite lectins have been cloned and characterized- In vitro- attachment to epithelia can be inhibited by adding free galactose to the medium

Attachment uses the secretion of a pore-forming peptide that is involved in lysing the host cell membrane

Infections can cause increase in IL-8 in the surrounding epithelial cells- this brings more lymphocytes to the area, these can serve as target cells for the parasite

Immune mechanisms:

Protective antibodies

Antibodies in animal models directed to the carbohydrate-binding lectins of the parasite

Cell mediated killing of parasites via reactive oxygen molecules

Most infections are asymptomatic- produces carriers

Symptomatic- diarrhea, colic, flatulence, bloody stools, dysentery

In chronic condition- form ameboma (large granuloma containing cells, parasites, necrotic colon tissue)- detectable / palpable masses- sometimes misdiagnosed as malignancies

Colon becomes damaged- may stretch ulcerate, perforation, colon contents enter abdominal cavity- peritonitis

Perforated, inflamed bowel may adhere to abdominal wall, amebas may attach to and infect skin tissue- cutaneous amebiasis

Extraintestinal

Amoebas may damage mucosa down to circulation area of the submucosa- enter circulation- visit other tissues- especially liver

This may occur after symptomatic or asymptomatic colon invasion. About 50% of patients with liver abscesses have not complained about amebic colitis

Hepatic amebiasis is slow, progressive and insidiousGeneral fever, general pain in upper abdomen- may be diagnosed as tender liver of a swelling / mass on the liver

Lungs can be involved-effusion, pleurisy, lung abscess,

Can affect bronchia, pericardium, brain

Diagnosis

Microscopy- hard to tell E. histolytica and E. dispar apart

Detection of antigen (Elisa/Western) or PCR

Treatment

Several drugs- metronidazole

Often given with another drug to kill cysts

Prevent infection- heating or freezing kills cysts

Normal chlorination levels do not kill cysts

A cyst of Entamoeba histolytica.  This is a mature cyst and, therefore, contains four nuclei.  However, only two nuclei are clearly visible in this plane of focus; approximate size = 18 µm.

Entamoeba histolytica trophozoite.  The single nucleus with its central endosome and regularly distributed chromatin is visible; approximate size = 22 µm