Transcript Lecture_4_Jan_13_2015_protozoa and giardia

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 cyclediversity 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 fatcarbohydrate 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 cytokinesisproducing 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 arginineless 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
Described in 1800’s
Schaudinn 1903 depicted trophozoites and cysts and
promptly died from overwhelming amebiasis
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
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 tissuesespecially 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 insidious
General 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