AMEBAS AND CILIATES. Parasites can be studied by one being aware of the following relevant aspects of their parasite biology:

Transcription

1 1 LABORATORY 2 PARASITOLOGY NOTES AMEBAS AND CILIATES EUKARYOTIC PARASITES: Parasites can be studied by one being aware of the following relevant aspects of their parasite biology: 1. Distribution (where are they found primarily) 2. Morphology of each stage of life cycle 3. Transmission mechanism of entry into host 4. Niche in host (tissue predilection) 5. Reproduction 6. Virulence factors (mechanism of survival in host) (Covered in lecture) 7. Mechanisms of Pathogenesis in host (6 & 7 may be same/similar in some parasites.) 8. Diagnosis and Treatment (Rx will be covered in lecture) GENERAL CHARACTERISTICS OF PARASITIC PROTOZOA: 1. Single celled organisms 2. One nucleus Exceptions: Giardia lamblia Has two nuclei that are transcriptionally active 3. Organelles (are eukaryotic) 4. Means of locomotion: Flagellum, undulating membrane, or cilia 5. Aerobic and anaerobic metabolism represented in the protozoa 6. Energy source Host usually! MECHANISM OF ENTRY FOR PROTOZOA: three ways 1) Oral (eaten!!) 2) Sexual 3) Vector bites

2 2 NICHE IN HOST: -complex molecular environment which meets physiological, biochemical needs of parasite 1) Type of Host -Specific - parasite found in only one type of host -Nonspecific parasite found in many types of hosts -Zoonotic parasite will readily change hosts Zoonosis parasitic infection parasite transmitted from animal to man 2) Tissues within a host Specific some parasites extremely specific and will occupy only one specific niche in human host such as malaria in human RBC s General - some extremely general such as Toxoplasma - any old nucleated mammalian cell will serve as host niche REPRODUCTION 1. Definitive Host - harbors sexual stages - sexual reproduction occurs in this host By this definition, in malaria, Anopheles mosquito is definitive host not man Also not all genera of protozoa have sexual stages 2. Intermediate Host: - Required in life cycle of parasite - Parasite undergoes life cycle development in intermediate host -No sexual reproduction by parasite in this host! 3. Paratenic or Transport Host: - No development but parasite remains alive and infective - Enables parasite to enhance survival until it can reach appropriate host Possibly evolutionary link between intermediate and definitive host e.g. Owl is definitive host for certain species of Acanthocephala Intermediate host is an Insect that eat eggs passed in owl feces Owls don t eat insects, but shrews do. Shrews accumulate heavy load of infective stages of the acanthocephalan parasite without any effects. (parasite does not undergo any life cycle development in the shrew!) Owls do eat shrews and become infected with the parasite. Shrew is paratenic or transport host a link that the parasite is able to use to reach its definitive host and complete its life cycle. All life cycle development occurs in the owl and the insect.

3 3 4. Reservoir host: A host that sustains a parasite in an environment. This host is a source of infection and potential reinfection in humans. (There is some development in this host but usually the parasite has no effect on this host!) Reservoir host is not the same thing as an intermediate host!!! It is NOT REQUIRED in the life cycle of the parasite. Reservoir can also be normal host. Remember, a reservoir host maintains the organism in the environment. General Rule for Protozoa: asexual reproduction in host Allows one infective organism to result in many organisms within host!!! 1. Binary Fission one parasite divides producing two parasites 2. Multiple Fission one parasite divides to produce many -Repeated nuclear and organelle division occurs within parasite -Cytokinesis results in many more of the same stage -Schizogony: --Schizont daughter cells called Merozoites --Each merozoite repeats process -Merogony: --Merozoites producing more merozoites is schizogony and is called merogony -Gametogony: --Merozoites produce gametocytes instead of more merozoites MECHANISMS OF SURVIVAL (virulence factors) mechanisms that parasites use to survive in the host: Examples: 1. Inhibit host defenses 2. Undergo antigenic variation 3. Coat self with host proteins as camouflage

4 4 AMEBAS AND CILIATES: Kingdom: Protista Phylum: Sarcodina ( amebas - protoplasmic flow with and without discrete pseudopodia) Class: Lobosea (pseudopodia are lobose finger shaped with a round tip) Order: Amoebida (typically uninucleate, mitochondria, no flagellate stage, asexual) Family: Entamoebidae Entamoeba histolytica (amebic dysentery) Entamoeba coli Entamoeba gingivalis Endolimax nana Iodameba buetschlii Order: Schizopyrenida (typically - uninucleate, flagellate stages) Family: Schizopyrenidae Naegleria fowleri (free-living opportunist) Family: Hartmanelllidae Acanthamoeba spp (free-living opportunist) Phylum: Ciliophora (ciliates) Class: Litostomatea Order: Vestibuliferida Balantidium coli (balantidiasis) PHYLUM SARCODINA: General Comments amebas: -Single celled -Motile; crawl around by rapidly extending and retracting pseudopodia -Phagocytic -Most are free-living in soil or water -Only a few are parasitic and most of these are commensals -Asexual reproduction SOME TERMS: Chromatoidal bars (chromatid bodies): crystalline-like condensation of ribosomes formed as trophozoite encysts Amebulae: metacystic trophozoites small but basically morphologically the same as the large trophozoites Endosome/karyosome: like a nucleolus in nucleus (size and location are diagnostic feature in ameba)

5 5 Chromatin granules: This is DNA; see on inner edge of nuclear membrane (size of granules may help differentiate between E histolytica and commensals) Food vacuoles: seen as round, light colored spots in trophozoites; are fluid-filled Commensals: non-pathogenic parasitic organisms Family Entamoebidae: parasites in digestive tract of man and other animals most are commensals but some are serious parasites species differentiated primarily on basis of nuclear structure Life cycle stages: 1. Trophozoite - nucleus, granules, food vacuoles in cytoplasm -crawling and feeding 2. Pre-cyst - trophozoite has rounded up and begun to secrete a tough, hyaline cyst wall and begun to form chromatoidal bars 3. Cyst -nucleus divides to form 4 nuclei - quadrinucleate - the mature infective stage 4. Metacystic trophozoites amebulae - after excysting in the small intestine, cytoplasm and nuclei divide to form 8 small trophozoites (amebulae) Entamoeba histolytica (lacks mitochondria and golgi) distribution: world-wide a serious pathogenic ameba -causes amebiasis and amoebic dysentery Transmission: oral: fecal contamination of food and water (can occur also mechanically via roaches and flies) Infective stage: Cyst with 4 nuclei Niche in host: Large intestine Life cycle: Direct Trophozoite in large intestine rounds up to form Pre-cyst which starts to secrete cyst wall. Cyst: when cyst wall is formed the stage is now a cyst. Within the cyst, the nucleus divides to four 4 nuclei. The cyst has now matured to become the infective stage, a quadrinucleated cyst. Large numbers of infective cysts are passed with feces. Cysts can remain viable and infective for weeks to months in a warm, moist environment. When ingested, the cyst passed through to the small intestine where it begins to excyst. The four nuclei and the cytoplasm from the cyst divide to form 8 metacystic trophozoites. also called amebulae which are carried to the large intestine by peristalsis. There they colonize the epithelium by attaching to epithelial cells. In the large intestine, the trophozoites can be active and crawl around via pseudopodia. The trophozoites feed, grow to the mature trophozoite size and divide by binary fission frequently. They feed on

6 6 starches and mucous secretions of large intestinal epithelium and engulf and hydrolyze mucosal cells and red blood cells when present. A diagnostic feature for E. histolytica is the presence of red blood cells in food vacuoles within the trophozoites. Normal infection: Intestinal amebiasis only large intestine is involved. Engulfment and lysis of epithelial cells creates ulcers in large intestine lining and results in colicky pain and protracted diarrhea lasting more than a few days. Dysentery - bloody diarrhea can be fatal. Ulcers are eroded deep into the vascular part of the mucosa of the large intestine resulting in the bloody diarrhea characteristic of dysentery. Amebomas: palpable masses which are composed of ameba, necrotic colon tissue and eosinophils. Can be seen in chronic intestinal amebiasis. asymptomatic carriers infected individuals who shed infective cysts without showing any clinical signs of disease! Severity of disease depends upon strain of E. histolytica, immune response of host and size of infective dose of cysts. Extra-intestinal amebiasis: Invasive ameba erode ulcers deep into the intestinal wall through the submucosa and gain access to circulatory system, disseminating throughout the body The most common extra-intestinal site is the liver followed by the lungs. Liver abcesses should always be checked for amebiasis. There are cases of liver amebiasis without any intestinal signs. CNS involvement is not common but has a high mortality rate when it does occur Extra-intestinal ameba are on a dead-end trip. They cannot leave the host nor form cysts outside of the intestinal lumen.

7 7 ingested RBCs (red arrow) nucleus (blue arrow) E. histolytica trophozoites E. histolytica trophozoites on slides: um -single nucleus with central endosome/karyosome -Chromatin granules, small and distributed peripherally around inner surface of nuclear membrane -a diagnostic characteristic: ingested RBC s -trophs stain dark on slides E. histolytica cysts E. histolytica cyst on slide: um - cigar-shaped chromatoidal bars(rounded ends) -4 nuclei with central endosome/karysome (early cyst may have only 1or 2 nuclei) -spherical on slides; nuclei stain dark; cyst wall won t stain

8 8 Your text divides the life cycle into five stages: trophozoite, pre-cyst, cyst, metacyst and metacystic trophozoites. A metacyst is simply the mature cyst that is the infective stage. It is quadrinucleate and may have no chromatoidal bars. As you can see by the diagram, the chromatoidal bars appear and then gradually disappear in the cyst stage. Entamoeba coli: the most common non-pathogenic species in the human colon can co-exist with E. histolytica in colon does not damage or feed upon host tissue (eats bacteria, protozoa, yeasts) Trophozoites are very similar to E. histolytica Cysts have 8 nuclei Life cycle: direct -- cysts out in feces; ingested orally (like E. histolytica)

9 9 E. coli trophozoite um eccentric endosome/karyosome in nucleus peripheral nuclear chromatin unevenly distributed difficult to differentiate from E. histolytica trophs E. coli cysts: um wide 8 nuclei with eccentric endosome/karyosome (over 4 seen is E. coli Can see 6 above) Chromatoidal body (bar) if present, is splintered appearance on ends Note: - to see endosome/karyosome, will require 100X oil immersion - look for spherical objects on slide nuclei will stain darker - cysts will be very round, trophs not necessarily round (see photos above for E. histolytica and E. coli) Entamoeba gingivalis Trophozoites only NO cysts Trophozoites identical to E. histolytica commensal in mouth (currently some controversy about non-pathogenic status) found in primates, dogs and cats Transmission: direct trophozoites via kiss, saliva, shared eating utensils

10 10 E. gingivalis slide -do not confuse with epithelial cells (big dark nucleus in epithelial cell is give away) -to distinguish from E. histolytic, E coli - sample source: E. gingivalis won t be in a fecal sample!! -food vacuoles containing bacteria, white blood cells, epithelial cells being digested (shed epithelial cells) and red blood cells if present (red arrow above) Iodameba buetschlii trophozoite cyst only species in this genus infects man, other primates and pigs most common ameba in pigs (probably original host species) low incidence in humans commensal in large intestine (cecal area primarily) Feeds on bacteria in gut Direct life cycle cysts out in feces

11 11 TROPHOZOITE (large endosome CYST in nucleus) I. buetschlii Trophozoite: comparatively large nucleus no chromatin granules at nuclear membrane glycogen vacuole large endosome (about ½ diameter of nucleus) food vacuoles 9-14 μm I buetschlii cyst: single nucleus (always close to vacuole) large endosome large glycogen vacuole (occasionally may be two present) 9-15μm (approximately same size as trophs) Endolimax nana Smallest of the amebas commensal large intestine Direct life cycle ingestion of cysts endosome, large, irregular and variable in shape nucleus with thin ring of chromatin at membrane size: <10μm

12 12 E. nana trophozoite Look at the size bars: these are very small ameba ORDER SCHIZOPYRENIDA Naegleria fowleri Found worldwide in soil and warm, stagnant fresh water (including unchlorinated swimming pools) Opportunist not a normal parasite of humans - is free-living and does not need a host in its life cycle -infection occurs when person swims or dives into water where biflagellated stage of ameba cycle are found -enter body through nose (probably forced in when diving), travel to brain via olfactory nerve and into the brain lysing their way through brain creating ulcerations in brain tissue. PAM primary amebic meningoencephalitis -most cases are fatal within 5-10 days post infection -Clinical symptoms include headache, fever, stiff neck, confusion, loss of coordination, coma and death. Infection is most common when temperature is high and water is warm -Positive note: infections are rare. -Prevention: Stay out of polluted and unchlorinated stagnant water.

13 13 N. fowleri trophozoites in brain tissue flagellum N fowleri trophozoite binucleate (preparing to divide) N. fowleri Biflagellated form (2 flagella) N. fowleri trophozoites uninucleate large ameba um in diameter

14 14 Acanthamoeba spp Distribution: worldwide -found everywhere: soil; water including freshwater, sea water, brackish water; airborne dust -free-living - does not need host in life cycle but is an opportunist! Transmission: cutaneous and inhalation -primary inoculation sites: skin, lungs, eye (associated with contact lens wear) --organisms have been isolated from nose and throat of healthy individuals indicating probably commonly inhaled without being infected Morphology: Trophozoites and cysts found in human hosts -Trophozoites small and may see spiky pseudopodia but probably not - Cysts have a crystal like shape (not round) At right: corneal biopsy Acanthamoeba trophozoite At left: corneal biopsy showing a cyst of Acanthamoeba

15 15 Clinical Disease: eye, skin or brain EYE: Ulcerative keratitis - can start with lesion in cornea (scratch) and dust or dirt getting into eye -85% of cases, however, associated with extended wear contact lens lack of sanitation for cleaning contact lens -washing in tap water (remember, they are in water and can be in water that comes in through your taps in your home!) -using non-sterile saline solutions -contaminating sterile lens cleaning solutions wearing your contacts while engaging in any water activity organisms and cysts get trapped by contact lens and chow down on your cornea Clinical signs: tearing and ocular pain squinting, conjunctivitis corneal opacity lesions deepen into corneal ulcers. Untreated, organisms will invade through corneal ulcer into eye all the way to the retina. Dx: corneal scrapings will find both trophozoites and cysts Rx: Antifungal and anti-amebic drugs Debride cornea to remove necrotic tissue Corneal opacity & ulceration Corneal transplant may be required to restore vision. BRAIN: GAE Granulomatous Amebic Encephalitis - This organism will invade the brain in immunocompromised individuals not immunocompetent individuals -slower onset compared to PAM -Ulcerative granulomas form in brain around parasites -Fatal Clinical Signs: headache, seizures, death Rx: none

16 16 PHYLUM CILIOPHORA (ciliates) General Comments on ciliates: Possess cilia (compound or simple) in at least one stage of life cycle Two kinds of nuclei: - Macronucleus -very large - Micronucleus Reproduce by transverse binary fission All have a direct life cycle Family Balantidiidae Balantidium coli very large ciliate (largest protozoan parasite in humans) μm only ciliate in humans (infection rate very low, only about 1%) distribution world-wide; especially in tropical regions many reservoir hosts especially pigs niche in host invade tissues of large intestine similar to E. histolytica in pathological effects, feeding upon host cells creating ulcers in large intestine can cause diarrhea, and dysentery which can be fatal if ulcer perforates intestinal wall Trophs move into large intestine lumen and encyst, with cysts passing out with feces Diagnosed by finding cysts in feces or organisms in biopsy of intestine Treatment tetracycline trophozoite

17 CYST μm - no cilia - dessication of feces encystment occurs - Encystment can occur outside the host. - Trophozoites passed in feces can survive and encyst outside of host --trophozoites can be infective if individual is malnourished or debilitated -fecal contamination of food or water -living trophs and cysts are yellow or greenish in color 17

18 18 AMEBA (characteristics used in ID of intestinal ameba) Trophozoites: 1. Motility 2. Cytoplasm -Coarsely granular -Finely granular 3. Nucleus -number (usually only one in trophozoite) -karyosome size and position -peripheral chromatin -present -absent -distribution and arrangement of granules Cysts: 1. Cytoplasm -inclusions (chromatid bodies, glycogen vacuoles, other elements) 2. Nucleus -Number -Karyosome size and position -Peripheral chromatin -presence -absence -distribution and arrangement of granules NOTE: size is not mentioned because it is variable especially in trophozoites and unreliable as a diagnostic criterion. For amebas, the nucleus is usually the single most important morphological characteristic for identification of stained specimens but always keep in mind that variation from the norm is common. Entamoeba histolytica may have a karyosome that is not small, nor centrally located. Points to Ponder: 1. Which ameba has no cyst stages? 2. Which ameba has cysts with splintery chromatoidal bars? 3. Which ameba has cysts with blunt chromatoidal bars? 4. What is the primary that parasitic amoebae are transmitted from host to host? 5. What are the exceptions to this general method of transmission? 6. How would you differentiate Entamoeba histolytica from Entamoeba coli?

19 19 HELPFUL HINTS for SCOPING OUT THE MICROSCOPE SLIDES: Be careful with the microscope. You will have to use it for the entire semester, so treat it gently and take good care of it. Follow the steps given to you in laboratory one on the care of microscopes. When looking for specimens on the slides, pay attention to the size of the organism so that you have a general idea of what you are looking for on the slide. You will have different types of prepared slides in this course: Stained tissue smears Stained blood smears Fecal smears Some stains used on slides include: iron hematoxylin, giemsa, trichrome, H & E (hematoxlyin & eosin), acid-fast You are probably most familiar with H&E-- purple nucleus and pink cytoplasm Trichrome is interesting: purple nucleus and green cytoplasm Iron hematoxylin stains nuclei, muscle striations blue-black to black Acid fast: parasites stain reddish and other structures counter stain bluish green Look for more than one specimen on a slide to positively identify the organism. Fecal smears are the most difficult. You have to learn to differentiate between the pseudoparasites and other garbage in the feces. Fecal smears typically have several layers focus on one layer and search. If negative, focus on another layer and repeat your efforts. If you need help, ask. Ask for verification. Make sure you are looking at what you think you are looking at. The accompanying atlas by John Sullivan is very good but it is no substitute for sitting at the microscope and learning to recognize the organisms on the slides used in this course. Suggestion: You may want to sketch the organisms at which you are looking. It will help you remember their morphology under the scope.