Biological Sciences 318 - Parasitology Lab Apicomplexa

1. Biological Sciences 318 - Parasitology Lab Apicomplexa

2. Apicomplexa Kingdom II Euprotista Phylum Apicomplexa Classes (& Genus): Gregarina (Monocystis), Coccidia (Eimeria, Sarcocystis, Toxoplasma, Haemogregarina), Haemosporidia (Plasmodium, Haemoproteus),Piroplasmea, Perkinsea Protozoa Ciliophora Apicomplexa Sarcomastigophora Gregarina Coccidia Haemosporidia

3. Apicomplexa Apical complex: • Polar rings (one or more) • Conoid • Rhoptries (cell penetration and nutrient transport) • Micronemes (attachment of parasite to host cell) • Subpellicular microtubules • Dense granules • (containing proteins; released after invasion to • function in calcium binding and formation of cysts) • Diverse group • All are Parasitic - endoparasitic • Most have complex life cycles • Many have a spore stage • Exquisitely evolved interaction with their host • Associations range from avirulent to virulent pathogens • No obvious external organelles involved in locomotion • No cilia or flagella, except for gametes • Feeding: Osmotrophy, Phagotrophy (Micropore/Cytostome) • • Asexual (Schizogonyor Endopolyogeny) and sexual reproduction (Syngamy, Conjugation). • • Include parasites with huge impact on human and animal health.

4. Apicomplexa Life cycles: Asexual (Schizogony or Endopolyogeny) and sexual (Syngamy) development 4 general components Sporogony: the product of a large number of sporozoites Merogony: production of merozoites Gamogony: formation of gametes Syngamy: fertilization of gametes to produce a zygote The sporont, meront, and gamont multiply asexually by schizogony [Schizogony: multiple nuclear divisions and then plasmotomy]

5. Class Gregarina: Genus Monocystis • some elements of AC converted into holdfast organelles (attachment to host cell) • Hosts: invertebrates primarily annelids (earthworm) and arthropods • no schizogonic phase (trophozoite instead merogony) • sporozoites form trophozoites in sperm mother cell of earthworm Life cycle: direct Slide 1.Troph attached to cell 2.Syzygy (2 Trophs associate in cyst) 3.Gamogony 4.Syngamy 5.Sporogony 6.Gametocyst with Oocysts each with 8 Sporozoites

6. Class Coccidia Intestinal Coccidia Eimeria, Isospora, Cyclospora, Cryptosporidium • Eimeria spp. • Intestinal coccidiosis • great economic importance in domestic animals • Every species of mammal has 2 to a dozen species of Coccidia! • Host: intracellular parasites (principally vertebrates) • Life cycle: complex; include 3 sequential stages endogenous merogony, gamogony followed by sporogony (exogenous) • Transmission: Ingestion of resistant Oocyst • Pathology: Diarrhea (watery, bloody), sloughing • of epithelium, cell death

7. Class Coccidia Intestinal Coccidia Eimeria, Isospora, Cyclospora, Cryptosporidium • Order Eimeriidae • Homoxenous (direct life cycle) • Merogony, gamogony and the formation of oocysts occurs within the same host. • Oocysts leave the host via the feces, and are unsporulated (undeveloped, non-infective). •  Exogenous Sporogony •  Sporulated oocysts of Eimeria contain 4 sporocysts, each with 2 sporozoites. • Eimeria stiedae is a parasite of rabbits • development in the bile ducts of liver • Transmission: ingestion of oocysts. • Sporozoites break out, travel to bile duct • Penetrate epithelial cells. • Undergo schizogony forming a schizont • Schizont produces merozoites • Some merozoites undergo gamogony • Syngamy • Oocyst

8. Class Coccidia Intestinal Coccidia Eimeria, Isospora, Cyclospora, Cryptosporidium Order Eimeriidae Slide: Liver tissue section infected with Eimeria stiedae. Trophozoites are round inclusions in the biliary epithelium. Macrogametocytes are large oval bodies with peripheral red-staining granules. Microgametocytes are few in number and more diffusely stained than macrogametocytes. Schizonts (contain 6-20 banana-shaped merozoites).

9. Class Coccidia Extraintestinal Coccidia (tissue Coccidia) Toxoplasma, Sarcocystis, Neospora • Large group of organisms important to humans and animals • Isosporoid oocyst: 2 Sporocyst with each 4 Sporozoites • Two host life cycles •  obligate for Sarcocystis •  facultative for Toxoplasma • Definitive Host (carnivore or omnivore) • adult forms • sexual reproduction • usually in intestinal tract • Intermediate Host (herbivore) • immature forms • asexual reproduction • usually extra intestinal (blood vessels, liver, muscle, brain)

10. Class Coccidia Extraintestinal Coccidia (tissue Coccidia) Toxoplasma, Sarcocystis, Neospora • Sarcocystis spp. • Sarcocystosis • Hosts • Definitive: canids (intestinal tissue) • Intermediate: domestic ox, sheep, ducks (somatic muscle) • Transmission • Definitive: prey-predator (ingestion of flesh) • Intermediate: Ingestion of Oocyst (intermediate host) • Two host indirect life cycle • Pathology • Definitive: not very pathogenic • Intermediate: brain, muscle, and kidney tissues • may be damaged by infection. • Clinical signs: loss of appetite, fever, weight loss, anemia, • and death in severe in infections. Slide

11. Class Coccidia Extraintestinal Coccidia (tissue Coccidia) Toxoplasma, Sarcocystis, Neospora • Toxoplasma gondii • Toxoplasmosis • Hosts (low host specificity) • Definitive: Felines (intestinal tissue) • Intermediate: almost any mammal, bird (most nucleated cell types) • Transmission • Definitive: prey-predator (ingestion of flesh) • Intermediate: Ingestion of oocyst (cat feces) • Ingestion of bradyzoites (raw meat) • Congenital • Two host indirect life cycle; intermediate host not mandatory! • Pathology • In most humans infected with Toxoplasma, the disease is asymptomatic.  • However, under some conditions, toxoplasmosis can cause serious pathology, including • hepatitis, pneumonia, blindness, and severe neurological disorders. •  immuno-compromised individuals (AIDS)

12. Class Coccidia Extraintestinal Coccidia (tissue Coccidia) Toxoplasma, Sarcocystis, Neospora • Two host indirect life cycle; • intermediate host not mandatory! • Extraintestinal phase (intermediate host) • Intestinal phase (definitive host)

13. Intestinal coccidia: Eimeria, Isospora, Cryptosporidium Genetically programmed number of asexual cycles- sexual reproduction-oocysts- exit with feces Extra Intestinal coccidia: Toxoplasma, Sarcocystis Genetically programmed number of asexual cycles- sexual reproduction-oocysts- exit with feces some stages enter muscle or other tissues- transmitted via predation

14. Class Heamosporidia Genus Plasmodium • This group includes the most pathogenic parasites of man. • Cosmopolitan disease in tropical and sub tropical regions of the world • Most important vector-borne disease • Romans called it “mala aria”- disease emanating from the swamps • The causal agent only identified in 1897 • 40% of the world population is at risk for contracting malaria. • It occurs in over 90 countries. • 500 million new cases and 2-3 million deaths annually. • Problems with vector resistance to insecticides, Plasmodium resistance to drugs, no vaccine have made malaria an excellent example of a re-emerging infectious disease.

15. Class Heamosporidia Genus Plasmodium • Human malaria parasites belong to one of four species • Plasmodium vivax benign tertian malaria, vivax malaria • Plasmodium falciparum malignant tertian malaria, falciparum malaria • Plasmodium ovale tertian malaria, ovale malaria • Plasmodium malariaequartan malaria, malariae malaria • Cosmopolitan disease in tropical and sub tropical regions of the world • It occurs in over 90 countries. • 500 million new cases and 2-3 million deaths annually. • Most deaths occur among young children in tropical Africa who are infected with P. falciparum. • .

16. Class Heamosporidia Genus Plasmodium Life cycle Two host indirect life cycle (Vector required for completion) Definitive Host: Mosquito; sexual cycle; Maturation of gametes, fertilization, Sporogony Intermediate Host: Human; asexual cycle; Merogony, Gamogony 3 Cycles: Sporogonic cycle, Exo-erythrocytic cycle (liver cells), Erythrocytic cycle (RBC)

17. Class Heamosporidia Genus Plasmodium Malaria • Symptoms of malaria include fever and flu-like illness, including shaking chills, headache, • muscle aches, and tiredness. Nausea, vomiting, and diarrhea may also occur. • Malaria may cause anemia and jaundice (yellow coloring of the skin and eyes) because of the • loss of red blood cells. Infection with one type of malaria, P. falciparum, if not promptly treated, • may cause kidney failure, seizures, mental confusion, coma, and death. • For most people, symptoms begin 10 days to 4 weeks after infection, although a person may feel • ill as early as 8 days or up to 1 year later. • Two kinds of malaria, P. vivax and P. ovale, can create problems later: some parasites can rest in the liver for several months to 40 years after a person is bitten by an infected mosquito, and can cause disease.

18. Class Heamosporidia Genus Plasmodium Slide: Blood smear of Plasmodium vivax: Plasmodium vivax can be recognized by its variable ring stage. Schizonts contain about 16 merozoites and the infected cell is enlarged and contains Schuffner’s dots. The disease caused by this Plasmodium is mild and known as benign tertian malaria(fever paroxysms typically every 48 hours) Schizont Ring stage Schizont with Schuffer’s dots

19. Class Heamosporidia Genus Plasmodium Slide:Blood smear of Plasmodium falciparum: has a very neat ring stage trophozoite. Multiply infected cells are common. Schizonts are rare in the peripheral blood. Gametocytes are crescent shaped. The disease caused by this organism is severe and known as malignant tertian malaria(fever paroxysms every 48 hours). It is this species that kills the vast majority of humans that die of malaria. Ring stage Schizont Macrogametocyte

20. Class Heamosporidia Genus Plasmodium Slide: Human malaria in liver tissue: Hemozoin (digested hemoglobin) deposited in the cells of the spleen in a human infected with malaria.  Slide: Sporozoite human malaria

21. Genus Haemoproteus & Haemogregarina Genus: Haemoproteus Parasites of birds Similar life cycles to Plasmodium (but: no asexual stages in circulating blood cells; they remain in the tissues). The gamonts remain in the erythrocytes. . Slide:Haemoproteus columbiae gametocytes. This preparation is a blood smear collected from birds. Note that bird erythrocytes are nucleated. You will find Haemoproteus gametocytes in the cytoplasm of the red blood cells. Genus: Haemogregarina are parasites of the blood of frogs, lizards and turtles, and are transmitted by leeches or mites. Related species occur in fish. Slide: Haemogregarines gametocytes in frog blood.

22. Monocystis sp. Eimeria sp. Haemogregarina sp. Plasmodium sp.

23. Learning Objectives Vocabulary Apical complex Schizogony Syngamy Sporogony Merogony Gamogony Oocyst Sporozoite Meront/Trophozoite Merozoite Microgametocyte Macrogametocyte Zygote Bradyzoite Sarcocyst Tachyzoite Exoerythrocytic cycle Erythrocytic cycle Sporogonic cycle Ring-form trophozoite Ookinete Schuffner’s dots Hemozoin • 1. Phylum Apicomplexa • - General characteristics • - Apical complex!! • - 4 general components of Apicomplexan development • 2. Class Gregarina – Monocystis spp. • Visual Id – common cysts, oocysts, sporozoite • Trophozoite stages feeding on sperm • There is no schizogonic phase • Host + tissue infected • 3. Class Coccidia – Eimeria stiedae • Host, tissues infected, transmission • Life cycle • Visual id trophozoite, macrogametocyte, microgametocyte, liver/bile ducts • 4. Class Coccidia – Genus Sarcocystis • - Host, tissues, life cycle, transmission • - Visual id • 5. Class Coccidia – Genus Toxoplasma • Life cycle, transmission, hosts, tissues infected • Intestinal vs. extraintestinal phase (where they occur, in what species…) • Pathogenecity • Visual id - tachyzoites • 6. Class Haemosporidia – Genus Plasmodium • Life cycle – what is definitive host • Transmission, • Visual id different stages and how they fit in life cycle • Pathology vivax and falciparum • Hemozoin in liver tissue • 7. Genus Haemoproteus • Visual id gametocytes • Life cycle, host , transmission, etc. • 8. Genus Haemogregarina • - Visual id, host, transmission