KINGDOM: PROTISTA SUBKINGDOM: PROTOZOA (“FIRST ANIMAL”) CHARACTERISTICS OF PROTOZOA 1. Unicellular 2. Chemoheterotrophs (get their energy by breaking down organic matter). 3. Most ingest their food; thus, they have special structures for this. 4. Trophozoites: any stage in a protozoa’s life cycle which can ingest food. In practice it refers to the motile form (pseudopods, cilia, flagella). 5. Cyst: Non-motile form, protected by a membrane. This is usually the infective stage. Cysts have a thick cell wall that allows for survival in harsh environments better than the trophozoite form. Excystation: process of emergence of the trophozoite from the cyst. 7. Capable of reproduction A. Asexual: fission, budding, or schizogony (produces a large number of trophozoites) B. Sexual: conjugation PROTOZOA CYSTS • Cysts are not as resistant as a bacterial endospore. • You can kill cysts by boiling them. • They can live in the soil or water for months. • A cyst is not motile, so it is not trophozoic. • A cyst does not go and seek its nutrients or ingest food, but it can absorb nutrients. • It has no organelles to ingest food.

TERMS: Host Types • The definitive host is the one in which the parasite completes its sexual life cycle. • For instance, in Plasmodium, the definitive host is the tropical mosquito anopheles. • The intermediate host is the human.

Kingdom ProtistaSubkingdom: Protozoa Phylum: Sarcomastigophora Subphylum: Sarcodina (amoebas) Amoeba spp. Entamoeba histolytica Subphylum: Mastigophora (flagellates) Giardia lamblia Trichomonas vaginalis Trypanosoma spp. Phylum: Ciliata (ciliates) Paramecium spp Balandtidium coli Phylum: Apicomplexia (Sporozoa; non-motile obligate parasites) Plasmodium spp Toxoplasma gondii Cryptosporidium Phylum: Sarcomastigophora(amoebas and flagellates) • Amoebas (move by pseudopods) • Flagellates (move by flagella)

1 Subphylum: Sarcodina (amoebas) • Amoeba spp (free living; not parasitic) ◦ Nucleus ◦ Pseudopods (false foot) • Entamoeba histolytica ◦ Nucleus ◦ Food vacuoles ◦ Do you see a cyst (4 nuclei) or trophozoite (1 nucleus)?

Entamoeba histolytica Disease: Amoebiasis • This is a global disease that any traveler can get. As soon as you cross the border into Mexico, you are exposed to it. • Entamoeba histolytica consume red blood cells. • In a fresh diarrheal specimen, you can see RBCs in the cytoplasm of the amoebas.

Entamoeba histolytica • Entamoeba histolytica, as its name suggests, can actually bore through the enteric walls (histolysis = destroying tissue) and reach the blood stream. • From there, it can reach different vital organs of the human body, like the liver, lungs, brain, eyes etc. • A typical effect is a liver abscess caused by such migrating Entamoeba histolytica, which can be fatal. •

Entamoeba histolytica • Entamoeba histolytica infection can lead to amoebiasis or amoebic dysentery. Symptoms include dysentery (diarrhea), weight loss, fatigue, and abdominal pain. • It can be diagnosed by stool samples. Trophozoites should be seen in a fresh fecal smear and cysts in an ordinary stool sample. Treatment Metronidazole Diagnostic Features • Ingested RBC • Bull’s eye Karyosome Subphylum: Mastigophora (Flagellates) • Giardia lamblia (intestinal parasite) ◦ Nuclei ◦ Flagella ◦ Do you see a cyst (4 nuclei) or trophozoite (1 nuclei)? • Trichomonas vaginalis (urogenital parasite) ◦ Nucleus ◦ Flagella ◦ No cyst stage • Trypanosoma spp (blood parasite) ◦ Flagella (for movement) 2 ◦ Undulating membrane (for movement) ◦ Nucleus ◦ Kinetoplast (circular mass of DNA)

Naegleria fowleri  Found in the soil and in freshwater lakes in the Southern part of the United States during the summer.  Infections usually occur when it is hot for prolonged periods of time  It can also survive in inadequately chlorinated swimming pools or dirty tap water, and can infect the brain when someone gets water up the nose.  Causes primary amoebic meningoencephalitis (PAM)

Mastigophora: Flagellates Giardia lamblia Disease: Giardosis Trichomonas vaginalis Disease: Trichomoniasis (an STD) Trypanosoma brucei Disease: African Trypanosomiasis Sleeping sickness Trypanosoma cruzi Disease: American Trypanosomiasis Chaga’s disease

TERMS • Mastigote = flagella • Promastigote: has single flagella • Amastigote: has no flagella • Kinetoplast: round mass of circular DNA Giardia lamblia Disease: Giardosis • Cysts are resistant forms and are responsible for transmission of giardiasis. • Both cysts and trophozoites can be found in the feces. • Infection occurs by the ingestion of cysts in contaminated water, food (includes undercooked meat), or by the fecal-oral route. Giardia lamblia Trophozoite form: piroform (pear or teardrop shape), looks like a happy face. • Discovered by Anton Van Leuwenhoek when he examined his own feces when he had this infection. • You won’t see the flagella in lab because you need a special stain for that. • Cyst form: oval shaped. Nuclei looks like two eyes.

Giardosis • Abdominal pain • Diarrhea • Gas or bloating 3 • Headache • Loss of appetite • Low-grade fever • Nausea • Swollen or distended abdomen • Vomiting Trichomonas vaginalis Disease: Trichomoniasis • Trichomonas vaginalis resides in the female lower genital tract and the male urethra and prostate. • The parasite is a trophozoite only; it does not have a cyst form, and does not survive well in the external environment. • Trichomonas vaginalis is transmitted among humans, its only known host, primarily by sexual intercourse.

Trichomoniasis Usually asymptomatic in men, or mild itching In women, vagina is extremely pruritic, with vaginal odor and discharge. African Trypanosomiasis (African Sleeping Sickness) American Trypanosomiasis (Chaga’s Disease) Trypanosoma brucei Disease: African Trypanosomiasis “African Sleeping Sickness” • Trypanosomiasis has a biological vector, the tsetse (pronounced “set-see”) fly. • Wild animals may also be a reservoir (Zoonotic is when a disease is transmitted to animals as well as humans.) • The tsetse fly bites a human and injects the trypanomastigotes into the skin. • This causes a chancre (pronounced “shanker”), which is an ulcer on the skin. • Then it enters the lymphatic system. Trypanosomiasis • It is characterized by Winterbottom’s Sign: swelling of the cervical lymph nodes in the head and neck area. • CNS symptoms include a shuffling gait (like a stroke victim), slurred speech, and malaise (needing to sleep longer and longer each day). • They are also restless at night. • CNS symptoms • Shuffling gait • Slurred speech • Malaise (sleeping all day) • Treatment • Melarsoprol: which has dangerous side-effects like chemotherapy. This drug requires administration with a substance called ethylene glycol, which will break down regular plastic tubing, so the drug must be administered with special plastic iv tubing.

4 Trypanosoma brucei Trypomastigote stages are the only form found in patients. • Posterior kinetoplast • Centrally located nucleus • Undulating membrane • Anterior flagellum Trypanosoma cruzi Disease: American Trypanosomiasis “Chaga’s Disease” • A zoonotic disease (can infect animals) that can be transmitted to humans by blood-sucking bugs. • This organism is a little smaller than T. bruceii and has a larger kinetoplast.

“Chaga’s Disease” • This disease is NOT found in Africa. • This disease is also zoonotic; it can infect animals as well as humans. • The vector is a large bug called the “Kissing Bug”. • It is found in warm regions and crowded areas, especially in the cracks of adobe huts. • It comes out at night and crawls on a human while they sleep.

Trypanosoma cruzi Triatomine bug, Trypanosoma cruzi vector, defecating on the wound after taking a blood meal.

“Chaga’s Disease” • It prefers the lips because the blood supply is close to the surface. • It sucks the blood there, but they don’t transmit the organism this way. • When they suck the blood, they also defecate, and the organism is in the feces. • When the human wakes up to scratch the itch, feces get into the tiny wound. • This is a fecal blood route.

Romana’s sign • Swollen eye, seen in Chagra’s disease. • Symptoms include fever, anorexia, swollen lymph nodes, hepatosplenomegaly (enlarged liver and spleen), and myocarditis (inflammation of the heart), which usually causes death. • They also have megacolon (large colon) and megaesophagus (large esophagus). Leishmania donovani • Disease: Leishmaniasis • Vector-borne disease transmitted by sandflies.

Leishmania Life Cycle

5 • It starts out as a spindle-shaped, single flagellated cell called a promastigote (mastigote means flagella). • You can also see the nucleus and a kinetoplast (mass of circular DNA).

Leishmania rosette In prepared slides you can see promastigotes align their nose in a circle, called a rosette.

Leishmania Life Cycle • It reproduces in the gut of a female sandfly , and migrates to her proboscis (mouth part). • It is introduced into the human by her bite. • It then enters a macrophage and becomes intracellular . • Here, it loses its flagella and is now known as an amastigote .

Leishmaniasis • These amastigotes multiply in various organs including the spleen, liver, and lymph nodes. • Symptoms include hepatosplenomegaly, lymph adenopathy, fever, weight loss, and a decrease in all blood cells: WBC, RBC, and platelets. • The treatment is almost as bad as the disease because of the side effects. It is best to catch it early. Leishmania Life Cycle • The female sandflies inject the infective stage, promastigotes, during blood meals. • Macrophages phagocytize them and they transform into amastigotes. • Other sandflies become infected during blood meals when they ingest infected macrophages. • In the sandfly's midgut, the parasites differentiate into promastigotes, which multiply and migrate to the proboscis Sandfly This looks like a mosquito, except its body is hairy and the wings are feathery. Leishmaniasis • Geographic Distribution:More than 90 percent of the world's cases of visceral leishmaniasis are in India, Bangladesh, Nepal, Sudan, and Brazil. • Leishmaniasis is also found in Mexico, Central America, and South America, southern Europe, Asia, the Middle East, and Africa. Leishmaniasis There are three forms of Leishmaniasis: • Cutaneous • Mucocutaneus • Visceral Cutaneous Leishmaniasis • The disease is only at the site of the bite. • This form is seen in Texas, Mexico, Asia, and the Middle East (our Iraq troops are coming down with this form). • It manifests as a large, wet sore with raised edges. It looks like a volcano with weepy serum coming out of the center. • The wound is not contagious, just the sandfly bite. • Dogs can get this disease, too.

6 Leishmaniasis (mucocunateous) • This is when the disease located in the mucous membranes of the nose and mouth. • The most gruesome photos are of this form.

Leishmaniasis (visceral) This is the most serious form. It occurs especially in immunocompromised people, especially HIV patients. ◦ The amastagotes reproduce inside macrophages. ◦ Only T-cells can kill infected macrophages, but HIV is a disease that infects T-cells. ◦ This form is known as Kala Azar.

Phylum: Ciliata (ciliates) Paramecium spp (free living; non-parasitic) • Oral groove • Macronucleus • Food vacuole • Cilia Balantidium coli (intestinal parasite) • Cytostome • Contractile vacuoles • Cilia Balantidium coli Disease: Balantidiosis • The animal reservoir is the pig. Its geographical distribution is world-wide, wherever humans and pigs live nearby each other. • This is the only ciliated protozoan that causes disease in humans. Balantidiosis • This is almost identical to Entamoeba histolytica. • The cyst form is infective. • It has a thick wall to protect it from stomach acid. • It enters the human (and dogs) by ingestion of fecal contaminants on food, water, and hands. • In the trophozoite form, they reproduce in the large intestine, invade the colon wall, and cause ulcerations in the colon. • Like Entamoeba, it leaves a flask-shaped ulcer. • Symptoms include diarrhea and GI discomfort.

Balantidium coli Trophozoites characterized by: • Large size (40 µm to more than 70 µm). • Presence of cilia on the cell surface • A cytostome (where they take in food) • A bean shaped macronucleus which is often visible and a smaller, less conspicuous micronucleus.

APICOMPLEXA • Characteristics: has an organelle called an apical complex which allows the organism to 7 attach to the host. • They all require a biological vector for transmission (to get into the blood of the host). Organisms Plasmodium Disease: Malaria Toxoplasma gondii Disease: toxoplasmosis Cryptosporidium parvum Disease: Cryptosporidiosis

Phylum: Apicomplexia(Non-motile obligate parasites) Plasmodium spp (blood and liver parasite) • Erythrocyte • Leukocyte • Parasite • Identify the stage: • Hepatic stage: Merozoites • Blood stage: Ring stage trophozoites

Plasmodium spp Disease: Malaria • 200-300 million infections a year • 2-3 million deaths a year • Affects mostly young people and teenagers • 2/3 of the cases are in Africa

Malaria • The term “malaria” comes from “mal air”, which means “bad air”. They used to think malaria was cause from the bad air of a foul-smelling swamp. Later it was discovered that the disease was caused by the protozoa inside the mosquito. • Sporozoites migrate into the salivary gland of the mosquito, and they are injected into the blood of the human. They immediately go to the liver. This is the only way to get malaria; you can’t get it from a blood transfusion because the transmission form is the sporozoite. They go right to the liver quickly to begin their next cycle.

Terms  Sporozoite: formed by sexual reproduction  Trophozoite: motile form of protozoa  Cyst: non-motile form of protozoa  Merozoite: Special name of the trophozoite form of Plasmodium after it hatches from a schizont.  Schizont: sack of Plasmodium organisms formed by sporozoites that have entered the liver. They metamorphasize into merozoites within the schizont, multiply until they rupture the sack and spread.  Schizogony: the process of forming a schizont.  Gametocyte: some merozoites differentiate into a male or female gamete. These are taken up 8 by the mosquito, and they combine into a zygote in the mosquito.

Plasmodium  The malaria parasite life cycle involves two hosts.  Sporogenic Stage  During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host.  Hepatic Stage  Sporozoites infect liver cells, form schizonts, metamorphasize into merozoites and multiply until the schizont ruptures and releases the merozoites into the bloodstream.  Blood Stage  Merozoites infect red blood cells  The parasites undergo asexual multiplication in the erythrocytes.  The ring stage trophozoites form another schizont, which ruptures, releasing merozoites.  Some parasites differentiate into male and female gametocytes.  Blood stage parasites are responsible for the clinical manifestations of the disease.

Malaria Symptoms • Fever, chills, sweating, headaches, muscle pains • Severe complications (cerebral malaria, anemia, kidney failure) can result in death.

Plasmodium - Malaria endemic malaria has been eradicated from U.S. • occurs in developing countries • current problems with drug resistance by the protozoan and insecticide resistance by vector • estimated that 40% of world’s population is at risk of acquiring malaria

Malaria (the problem) ~3000 children die of malaria/day in Africa • 1946 the CDC was founded to combat malaria • millions of cases of malaria in the U.S. in 1930s • in the U.S. the species of Anopheles that transmitted the parasite preferred to feed on cattle rather than humans • disease eradicated in U.S. in the 1950s • 2009 report in Emerging Infectious Disease that there are ~3,000 cases of non-endemic malaria in the U.S. each year

Plasmodium – Life Cycle  Mosquito (Anopheles) takes blood meal and injects sporozoites  sporozoites travel to liver cell (metamorphosis into trophozoite form, called merozoites)  They reproduce in the liver in a sack called a schizont (process is called schizogony) and 9 transform into merozoites. The y multiply rapidly until the sack ruptures, releasing the merozoites.  merozoites leave liver and enter RBCs (ring stage)  merozoites kill RBC and then:  infect more RBCs  to make more merozoites OR gametocytes  gametocytes taken up by mosquito  gametes join to form zygote, which develops into a sporozoite  sporozoites move to salivary glands

• once mosquito infected, infected for life • mosquito LC ~10 days (depending on temperature) • Anopheles bites many times

Human malaria 4 (major) species that infect humans • P. vivax • P. falciparum • P. malariae • P. ovale

Pathology • bursting RBCs – release of “toxins” • “sticky” RBCs can cause blockage in blood vessels (especially in lungs, kidney, spleen and CNS) • Sickle-cell patients resistant to falciparum malaria • Glucose-6-phosphate dehydrogenase deficiency patients have milder falciparum symptoms (parasite needs this enzyme)

Malaria paroxysm presents as acute febrile illness often followed by chills and rigors , then fever spikes (up to 40C/104F) and sweating severity depends on • species of Plasmodium • age of patient (more severe in children under 5) • other pathological conditions (superimposed bacterial infections, malnutrition, etc)

Malaria pathology (overview) symptoms due to: host response • fever – waste products of RBCs ◦ overproduction of cytokines (e.g. TNF) • paroxysm – sudden appearance of symptoms ◦ chills first, then fever, then back to normal until next attack (symptoms last ~8-12 hours)

10 ◦ falciparum has no stage where patient feels good anemia ◦ destruction of RBCs; cannot recycle iron fast enough ◦ infected cells can stick to un-infected RBCs which are then lysed ◦ ↓ RBC production by bone marrow (TNF ↓ erythropoietin) • Cerebral malaria - P. falciparum - ~10% of cases ◦ thought to be due to infected RBCs sticking to endothelial cells (including in the CNS) • Relapse (P. vivax/P. ovale) and Recrudescence (P. malariae)

Treatment Quinine Synthetic drugs: chloroquine, primaquine, etc multi-drug resistance (P. falciparum) vaccination • difficult because infection doesn’t provide effective life-long immunity • organisms shed protein coats • different antigens during infectious cycle

Anopheles Mosquitoes Female mosquitoes need blood meals to nourish their eggs. Toxoplasma gondii Disease: Toxoplasmosis • Infects most species of warm blooded animals, including humans. • Cats are the only known definitive hosts for the sexual stages of T. gondii and thus are the main reservoirs of infection. • Cats become infected by eating infected wild animals (e.g. birds) • Tissue cysts or oocysts are excreted in the feces. • Oocysts can survive in the environment for several months and are remarkably resistant to disinfectants. • Pregnant women who clean the litter box can catch the mild disease, but the fetus has severe symptoms. Toxplasmosis • Most primary infections produce no symptoms. The time between exposure to the parasite and symptom development is 1 - 2 weeks. The disease can affect the brain, lung, heart, eyes, or liver. • Symptoms are mild illness with fever, muscle pain, sore throat, headache, enlarged lymph nodes. • This organism prefers nerve tissue, so it travels to the CNS. • Pregnant women should avoid cleaning cat litter boxes, because the fetus can develop mental retardation, blindness and epilepsy, and stillbirths. Toxplasmosis Cyst Cyst in brain tissue Toxplasmosis Trophozoites Trophozoites

Cryptosporidium parvum 11 Disease: Cryptosporidiosis • Spread through the fecal-oral route, often through contaminated water • Causes self-limiting diarrhea in people with intact immune systems. • In immunocompromised individuals (such as AIDS patients), symptoms are severe and often fatal. AIDS patients have been known to have 50 stools a day, with tremendous water and weight loss. In these patients, the disease may persist for years.

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