VETERINARY PARASITOLOGY VPTH603
LABORATORY
last updated: 08/16/2005
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VETERINARY PARASITOLOGY VPTH603 last updated: 08/16/2005 |
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Laboratory #10 The Protozoa
Objectives: The protozoa are unicellular organisms that are classified on the basis of the organelles used for locomotion. In this laboratory, you will see many of the parasites that we have discussed in lecture. Some of these you will need to be able to identify because they are parasites commonly seen in practice. Others are shown to assist you in learning their life cycles. There are 4 parts to the laboratory, including (1) live/fresh materials; (2) wet preparations of fixed material; (3) demonstrations and AV; and (4) slides from the student slide box. The following live/fresh materials will be provided, each of which will demonstrate different life history stages: 1. Intestines of chickens infected with Eimeria tenella (schizonts and merozoites). 2. Hamster intestinal contents containing Giardia sp. and Trichomonas sp. (trophozoites) 3. Feces containing cysts for a ZnSO4 flotation. (cysts)
Ciliophora - The ciliates Balantidium coli -( Student Slides #55+56) This ciliate is a commensal of domestic animals (esp. swine). It can be pathogenic under some conditions. The trophozoite is found in the large intestine where it normally lives in the lumen, but it may invade the intestinal wall producing shallow ulcers. Diagnosis is made by finding the large cysts in the feces. In Slide #55 trophozoites can be seen in the lumen of a hamster or horse large intestine, in the epithelium. A few are deeper into the tissue. Note the large macronucleus, visible in some of the trophozoites, and the cilia on all the organisms (use your 40 X objective and low light to see the cilia).
In the stained fecal smear (Student Slide #56) note the cysts. They are spherical (40-60 µm) have a hyaline wall, and usually the large macronucleus can be seen within.
Sarcomastigophora - The amoebae and flagellates Entamoeba histolytica - DEMO This amoeba is one of the few pathogenic amoebae of veterinary importance. It is primarily a parasite of humans and primates, but can occasionally infect other hosts such as the dog. However, cysts are only passed in the feces of humans and primates, so they are the source of infection for domestic animals. Note the characteristic nucleus in the trophozoite. The trophozoite will have 1 nucleus and a feeding vacuole which may contain red blood cells.E. histolytica presents a diagnostic problem in dogs as only the trophozoite will be passed in the feces. Since a salt float would destroy this stage the only way to see them is to do a direct smear (staining the smear greatly improves your chances of identifying the amoeba).
Giardia sp. - DEMO, Wet prep. and ZnSO4 Float. (Foreyt, pg. 35)This flagellate is an inhabitant of the small intestine of mammals and birds. The cyst stage is usually found in the feces of infected animals, but in diarrhetic stools the trophozoites can sometimes be seen. To examine stools for the trophozoite you must make a thin direct smear and add a little saline to keep it moist (water may lyse the trophozoites and iodine would kill them making it impossible to see their characteristic motion). The cysts will float in standard salt solutions, but the salt solutions cause osmotic damage to the contents of the cysts. Therefore, a ZnSO4 solution (which is not saturated, thus, less osmotically damaging) is used for flotation. A) Wet mount - Take a drop of hamster small intestinal contents and place on a slide with a cover slip (no iodine). Look for the characteristic "Falling leaf motion" of the pear-shaped trophozoites (there will be some fast moving Trichomonas in this sample in addition to the less highly motile Giardia). To see a short video clip CLICK HERE (this is a very, very large file, do not download over a phone modem). B) ZnSO4 Flotation - take a small sample of dog feces and place it through a sieve into a tube full of ZnSO4 solution. Pour the slurry back into a tube and centrifuge for 1 min. Remove the cysts, stain them with a drop of iodine and examine them under the 40X objective. The cysts are small (10-15 µm - just visible at 10X), oval in shape, and contain remnants of the trophozoite organelles (usually the remains of the axostyle can be seen cutting across the long axis of the cyst). (If you have trouble finding Giardia cysts, there are several prepared slides on the middle bench, please return them after you use them.)
Trichomonas spp. - Student Slide #42, Wet Mount.Flagellates of this genus are found in the digestive tract or reproductive tract (T. foetus) of mammals and birds. In most cases, Trichomonas spp. are normal inhabitants of the digestive tract (esp. the cecum) and are generally considered non-pathogenic. There have been reports of diarrhea in which large numbers of Trichomonas sp. have been seen, but these reports haven’t shown that these flagellates were responsible for the condition. T. foetus is the only proven pathogen of veterinary importance and will infect cattle and cats. A) Wet Mount - make a wet mount of hamster cecal contents and look for Trichomonas muris. At 40X and low light you should be able to see the undulating membrane as a flickering wave on the body of the trophozoite. These flagellates don’t form cysts, so the diagnosis depends on identifying the trophozoite. Note how the movement of this organism differs from Giardia. To play a video clip of this parasite moving in a wet mount CLICK HERE (Note: Very, very large avi file do not download over a modem.)
Trypanosoma brucei - Student Slide #39 Trypanosomes are extremely important parasites of domestic animals in Africa and South America. In North America, they are common parasites of birds, in which they may occasionally be pathogenic. T. brucei is used in this lab to show the typical morphology of the trypanosomes. This African flagellate is found in the blood and cerebrospinal fluid of mammals. Use your oil lens and observe the trypanosome with its undulating membrane, single anterior flagellum and kinetoplast.
Leishmania donovani - Student Slide #41 Leishmania spp. are common parasites in tropical and sub-tropical areas of the world. They are found in rodents, dogs and humans. At VHUP, we have seen several cases of visceral Leishmaniasis in dogs that were brought to this country from the Mediterranean region and the CDC is currently investigating infections in foxhound kennels in the eastern United States. This parasite exists as a flagellated promastigote in the gut of a sand fly or as a non-flagellated amastigote in the macrophages of a mammal. L. donovani is found in the macrophages of the bone marrow, liver and spleen. Slide #41 (use your oil lens) is a spleen impression smear. This type of smear is made by cutting the organ and gently blotting on a paper towel to remove excess blood. The cut surface is then touched to a glass slide to leave the impression. The spleen cells rupture due to the excess surface tension and the cell nuclei and amastigotes are left behind. The cell nuclei stain as purple blobs and smears, while the amastigotes show up as small (2-3 µm) circles with a nucleus and kinetoplast.
Amastigote (arrow) in lymph node impression smear. Drawing of an amastigote.
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Copyright University of Pennsylvania 2005 Comments or Questions contact Dr.
Thomas Nolan at
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