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Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information Part I Methods to study primate–parasite interactions © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information 1 Collection methods and diagnostic procedures for primate parasitology ellis c. greiner and antoinette m c intosh Photograph by Jessica Rothman Introduction A great deal of energy has been expended on studying parasites of free-ranging primates. Many of these studies have been confined to fecal surveys as it is both difficult and not practical to gain knowledge of parasites in these hosts by using the older and normal method of studying the parasites that are recovered at Primate Parasite Ecology. The Dynamics and Study of Host–Parasite Relationships, ed. Michael A. Huffman and Colin A. Chapman. Published by Cambridge University Press. C Cambridge University Press 2009. 3 © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information 4 Primate Parasite Ecology necropsy. Thus, this chapter is designed to aid researchers in being as productive and efficient as possible in gathering information that is most useful for the conservation of these interesting creatures. The type of information obtainable depends upon the restrictions placed on the collection and preservation of the samples to be examined. If the primates are darted and anesthetized, then feces, blood, and ectoparasites may be collected. If the primates are not alive, then parasites and specimens could be recovered from various organs and tissues at necropsy. We need to be opportunistic with such endeavors as we cannot make the contributions with only feces that we can from recovery of parasites at necropsy. The eggs or worms found at necropsy have not been matched to the adult worm identifications in most cases as they have been with domestic animals species. Feces, therefore, is the only practical sample that can be collected and examined with reference to parasites in free-ranging primates for which capture is not an option. Some general references that might be useful for aiding in the detection and identification of primate parasites include those by Melvin & Brooke (1974), Anon. (1979), Zajac & Conboy (2006), and Garcia (2007). A flow chart of potential diagnostic procedures is depicted in Figure 1.1. Collection of fecal and blood samples precautions Because primate blood and feces are potential sources of organisms infectious to humans, personal safety of individuals collecting these samples in the field is imperative and can be achieved by following “universal precautions.” “Universal precautions,” as defined by the Centers for Disease Control (CDC), “are a set of precautions designed to prevent transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other bloodborne pathogens when providing first aid or healthcare. Under universal precautions, blood and certain body fluids of all patients are considered potentially infectious for HIV, HBV and other bloodborne pathogens” (CDC website). Basically, this means wearing gloves and protective clothing, such as disposable aprons, thorough hand washing, proper disposal of needles, scalpels, syringes and any items contaminated with blood, mucous, feces, and other body fluids in appropriate leak-proof and puncture-resistant containers for disposal. Refer to OSHA (Occupational Safety and Health Act) regulations for specifics on “universal precautions” and recommendations and procedures to follow in case of animal bites, contaminated needle sticks, or wounds or cuts becoming exposed to any of these fluids. Some points to consider in maintaining sample integrity for optimal parasite detection are: © Cambridge University Press www.cambridge.org © Cambridge University Press - Piroplasms - Mites SKIN SCRAPING - Lice - Mites - Ticks PICK ECTOPARASITES - Lice - Mites BRUSH FUR Figure 1.1. Flow chart of diagnostic procedures. GIARDIA ELISA (IDEXX SNAP TM TEST) COPROCULTURE - Strongylate nematode larvae - Organ slide impressions Prepare blood smears and impression smears of solid organs Mount lice and mites in Hoyer’s medium on microscope slides or submit to taxonomist Ticks and lice examine wet Fix arthropod in 70% ethanol ARTHROPODS idia Place in glacial acetic acid, store in glycerin alcohol, temporarily mount in lactophenol Relax flatworms in tap water for a few hours then fix in AFA Allow acanthocephalans to relax overnight in tap water to evert the proboscis, fix in AFA HELMINTHS - Fecal smear for Cr - Sedimentation yptospor - Flotation FIX IN FORMALIN FIX IN PVA - Perform Trichrome stain for amoebae and gut flagellates FECAL EXAM POSTMORTEM EXAM Fix tissue in formalin for histological exams NECROPSY PARASITE RECOVERY - Frozen specimens - Frozen feces - Histological sections BIOPSY - Drop of blood on filter paper or FTA elute card MOLECULAR DIAGNOSTICS INTEGUMENT EXAM - Trypanosomes - Microfilariae - Malarial parasites GEIMSA STAIN BLOOD SMEAR BAERMANN PROCEDURE - Nematode larvae - Adult pinworms FECAL SMEAR - Acid fast stain for Cr oocysts - Protozoan cysts yptosporlarvae - Nematode - Coccidian oocysts - Acanthocephalan eggs - Cestode eggs FLOTATION - Nematode eggs - All parasites listed under flotation - Fluke eggs SEDIMENTATION - Motile protozoa idia DIRECT SMEAR FECAL EXAMS ANTEMORTEM EXAM PARASITE DIAGNOSIS Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information 6 Primate Parasite Ecology 1. Avoid contamination of sample with water or urine “because water can be contaminated with free-living organisms that can be mistaken for (human) parasites” (Garcia, 2007). 2. Loss of motility of protozoa may occur if sample is contaminated by urine. 3. Time frames for examination or fixation (recommended by Garcia, 2007): a. Liquid feces: Examine or preserve within 30 minutes of passage (trophozoites). b. Soft feces: Examine or preserve within 1 hour of passage (trophozoites and cysts). c. Formed feces: Examine or preserve within 24 hours of passage (cysts). 4. If quantity allows for attempt at larval culture, do not refrigerate this portion of sample. Fecal exams Examination of feces is useful when attempting to gain information of the parasite fauna in a given primate. There are a number of procedures that could be used to detect the different groups of parasites. The number of exams that can be performed on one sample depends somewhat on the volume of feces obtained from each host. If at least 2 g of feces can be obtained, then the following procedures could be done: fecal flotation, fecal sedimentation, direct smear, a trichrome or iron hematoxylin stain for protozoa from fecal smears prepared after appropriate fixation, acid-fast stain for Cryptosporidium sp. on unfixed smears, and Baermann procedures for larval or tiny nematode recovery. How the samples are collected and fixed will depend on whether the examinations will be conducted in the field or sent away to a reference laboratory for processing or taken back into a laboratory setting. Because of the potential for the presence of infectious organisms, sample containers should be placed in plastic leak-proof bags before transport and the shipping container needs to be able to withstand the rigors of postal systems. Solutions for fecal procedures Saturated sodium nitrate Nitrate of soda (commercial grade fertilizer) 5 lb Hot tap water 1 gallon © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information Collection methods and diagnostic procedures 7 OR Sodium nitrate 400 g Hot water 1000 ml Stir ingredients in appropriate-sized container until dissolved. Specific gravity should be 1.20–1.25. The actual specific gravity should be measured with a hydrometer and recorded on the container when each batch of flotation solution is prepared. Sheather’s sugar Granulated sugar 454 g (1 lb) Tap water 355 ml (12 fluid oz) Liquefied phenol crystals or formaldehyde 6.7 ml (a preservative and mold inhibitor or simply store in the refrigerator) Dissolve sugar in hot water by stirring over a heat plate. After sugar is dissolved and the solution has cooled to room temperature, add liquefied phenol (or formaldehyde) or store in the refrigerator. The specific gravity should be 1.27 and this should be checked with a hydrometer and recorded on the container when each batch of flotation medium is prepared. Detergent solution for simple sedimentation Add 5 ml of liquid dish detergent into 4 liters of tap water. Avoid formation of bubbles when in use. Zinc sulfate for detection of Giardia sp. cysts ZnSO4 · 7H2 O 336 g Distilled water 1000 ml Mix and adjust solution to specific gravity of 1.18. While some parasitologists think they must use zinc sulfate in order to float cysts of this flagellate, sodium nitrate can be used with good results. 0.85% sodium chloride (normal saline) NaCl 8.50 g Distilled water 1000 ml This is used only for direct smear preparations looking for motility of gut inhabiting protozoa. © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information 8 Primate Parasite Ecology Saturated sodium chloride Water 1000 ml NaCl table grade Dissolve NaCl in water by stirring until there is a salt residue in the bottom of the container that will not go into solution. Procedures Fecal flotation Simple sodium nitrate flotation: for recovery of nematode and tapeworm eggs, coccidian oocysts, mites, and larval nematodes. Materials: Saturated sodium nitrate or ∗∗ FecasolTM specific gravity 1.2 15 ml conical centrifuge tubes and tube rack to hold them vertically (or FecalyzerTM ) Wooden tongue depressors or applicator sticks 50 ml paper cups or small disposable plastic containers Glass microscope slides and 22 × 22 mm coverslips Paper towels Compound microscope. Procedure using 15 ml centrifuge tubes: 1. Conduct steps 2 through 6 over paper towels. 2. Place 1–3 g of feces into the 50 ml container. 3. Add 15–20 ml sodium nitrate solution and mix well, thoroughly breaking up feces. 4. Place one layer of cheesecloth or gauze over the top of the container. Pour and strain the mixture into a 15 ml conical centrifuge tube (Figure 1.2) until you have formed a slight positive meniscus at the top. Set cover slip on top. The liquid should just touch the coverslip, but not spill over. Many eggs that have already floated to the top will be lost if you overfill the tube and spillage occurs when the coverslip is placed on the tube. 5. Let stand vertically for a minimum of 10 minutes (Figure 1.3). 6. Gently lift coverslip straight up without losing any fluid, and place it on a glass slide. © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information Figure 1.2. Setting up a flotation. Figure 1.3. Flotation running. © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information 10 Primate Parasite Ecology Figure 1.4. How to scan a slide systematically. Procedure using FecalyzerTM (strictly follow the directions that come with the FecalyzerTM ) The following is a basic summary of the procedure. This method assumes you have at least 2–4 g of feces to work with or you will be picking up any freshly dropped fecal samples. Using the apparatus, pick up a portion of the fecal sample in the bottom of the white center portion of the apparatus, and place it in the outer container that is half full of FecasolTM or sodium nitrate solution. Mix the sample well by turning and twisting the internal tube in the solution. Then press the white central portion down with sufficient pressure to seal the edges and fill the tube to the top forming a slight positive meniscus and place the coverslip on top being careful to avoid over filling and spilling. Let stand a minimum of 10 minutes (Figure 1.3). Remove coverslip by picking it straight up and place it on a glass microscope slide. Examine the entire area under the coverslip using a low power (10×) objective and adjust light intensity to give good contrast. Begin at one corner of the coverslip and scan cross to the opposite edge, then move down one field of view and scan back across to the opposite edge. Continue this method until the entire area under the coverslip is observed (Figure 1.4). If any suspicious objects are encountered or you want to observe details of eggs, cysts, or oocysts for identification and confirmation, move to the high-dry 40× objective (increase light slightly) and observe details and take measurements. This higher magnification is required to observe the characteristics of smaller protozoan cysts and oocysts in order to confirm their presence. Do not allow the slide to stand too long because the salt will begin to crystallize on the edges of the coverslip making the observation of any eggs or cysts in those areas difficult and may affect an accurate diagnosis if their presence is overlooked. © Cambridge University Press www.cambridge.org Cambridge University Press 978-0-521-87246-1 - Primate Parasite Ecology: The Dynamics and Study of Host-Parasite Relationships Edited by Michael A. Huffman and Colin A. Chapman Excerpt More information Collection methods and diagnostic procedures 11 Fecal sedimentation This method is used primarily to recover trematode and acanthocephalan eggs, but other helminth eggs, nematode larvae, mites, protozoan cysts, and oocysts will sediment as well. Materials: Detergent solution, cheesecloth, small plastic beaker, plastic 50 ml conical centrifuge tubes, appropriate size tube rack to hold tubes vertically, pipettes, glass slides, and coverslips. (Optional: 5% methyl green or 0.1% methylene blue stains if available), compound microscope, dissecting microscope if available. Procedure: 1. In a small beaker or cup, break up and mix 2–4 g of feces in a small amount of sedimentation solution and stir until uniform mixture is achieved (no large chunks). Continue adding detergent solution to an approximate volume of 50 ml avoiding the formation of bubbles. 2. Place one layer of cheesecloth over the beaker and pour mixture through cheesecloth into the 50 ml tube. If the tube is not full at this point, add more detergent solution to mixture by pouring it through the cheesecloth that is still covering the cup (the material trapped in the cheesecloth will be rinsed back into cup and possibly release more eggs into the solution), swirl to mix then continue by pouring through the cheesecloth from the cup into the tube until it is full or contains approximately 50 ml of filtered mixture. 3. Place the filled tube in rack and allow sedimentation to proceed for 5 minutes in a vertical position. 4. After a sedimentation time of 10 minutes, decant supernatant carefully and return to vertical position in one movement before any sediment reaches the lip of the tube or aspirate approximately 75% of the supernatant with a pipette to avoid mixing or losing the sediment at the bottom of the tube which contains the eggs. 5. Resuspend the sediment collected at the bottom of the tube by swirling or carefully tapping the bottom of the tube and refill tube with tap water by letting the water gently flow down the inside wall of the tube while avoiding the formation of bubbles. Note: Avoid formation of bubbles when adding water because eggs can be trapped in or adhere to detergent bubbles and will not descend during the next step and will be lost in the decanting process. 6. Allow to stand and sediment vertically for 5 minutes and decant as in step 4. © Cambridge University Press www.cambridge.org