1. Parasitology Sample Collection and Handling Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

2. Parasite Detection Parasites infect the oral cavity, esophagus, stomach, small and large intestine, and other internal organs Detected through microscopic examination of feces Life stage found in feces Eggs, oocysts, larvae, segments (tapeworms), and adult organisms External parasites Detected by skin scrapings, cellophane tape preparations, vacuuming, and brushing the coat Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 2

3. Collection of Fecal Samples Fresh samples are best If cannot be completed within a few hours Should be refrigerated or mixed with 10% formalin Older samples – the appearance of eggs, oocysts, or other life cycle stages may be altered due to parasite development Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 3

4. Small Animal Fecal Samples Several methods Owner collection immediately after defecation Stored in disposable container About 1 g (thimble-sized) is needed Labeled properly Collected directly from animal in hospital Gloved finger or fecal loop Fecal loop used for direct smear – small amount Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 4

5. Large Animal Fecal Samples Collected either directly from the animal’s rectum – glove tied and labeled Pooled sample from many animals comingled in one container Help get estimate of degree of infection with the group Label, species, pen, or group number Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 5

6. Samples Sample should be as fresh as possible Label – client name, date of collection, species, animal identification Examine samples within 24 hours Precautions Wear gloves and protective outerwear Prevents contamination of samples Ensures personal health Wash gloves frequently with soap and water Clean and disinfect work areas and equipment Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 6

7. Records Maintenance of good records are important Records should include: Identification information Procedures performed Results Should also include: Adequate history Clinical signs Duration of signs Medications given Environment Vaccinations Stocking density Number of animals affected Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 7

8. Equipment Microscope is the most reliable method of detection of parasitic infections Binocular microscope with x10, x40, and x100 objectives Calibrated ocular micrometer Stereo microscope is also helpful Glass slides View sample systematically x10 objective to start Many parasites in the same plane as air bubbles or at the edge of the coverslip Once a parasite is located, examine at higher magnification Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 8

9. Evaluation of Fecal Specimens The choice of test to be performed is dependent on clinical signs and patient history Begin with a gross examination of the sample Consistency Color Presence of blood Mucus Odor Adult parasites Foreign bodies Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 9

10. Direct Smear Simplest Requires minimal equipment Allows for visualization of trophozoite stages of protozoal parasites – Giardia Not adequate examination Disadvantages Small sample size Debris may be confused with parasitic material Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 10

11. Direct Smear (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 11

12. Fecal Flotation Based on differences in specific gravity of life cycle stages of parasites Flotation solutions have a higher specific gravity then common ova Ova float to surface Fluke eggs have a higher S.G. and sink Too high an S.G. creates a plug of debris that traps parasites, obscuring them from view Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 12

13. Fecal Flotation (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 13

14. Fecal Flotation (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 14

15. Centrifugal Flotation Similar to flotation except once mixed the sample is strained to remove excess debris Requires a tabletop centrifuge More sensitive than simple flotation Recovers more eggs and cysts in less time Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 15

16. Centrifugal Flotation (cont.) Giardia Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 16

17. Fecal Sedimentation Used when suspected ova are too large to be recovered with standard flotation Fluke ova Laborious technique Most fluke eggs do not float or they are distorted by flotation solutions, making them difficult to recognize Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 17

18. Fecal Sedimentation (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 18

19. Cellophane Tape Preparation Often used to recover ova from Oxyuris – pinworms and tapeworms Tape wrapped around a tongue depressor with adhesive side out Animal’s tail raised and tongue depressor pressed firmly against the anus Tape then removed and applied to a slide with a small amount of water Examine microscopically Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 19

20. Baermann Technique Used to recover larvae from fecal samples Requires the construction of a Baermann apparatus Large funnel, tubing, and collection tube Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 20

21. Baermann Technique (cont.) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 21

22. Baermann Technique (cont.) Routinely performed on feces of domestic animals when lungworm infections are suspected Fresh samples collected rectally In dogs and cats, Baermann technique is used when Strongyloides are suspected Zoonotic potential Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 22

23. Miscellaneous Fecal Examinations Some parasites produce intestinal bleeding Evident by frank blood in feces or darkened feces Some intestinal bleeding can only be identified with chemical testing Occult blood testing Many kits available Identify the presence of hemoglobin Vomitus can also aid in diagnosis of parasites Toxocara canis Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 23

24. Miscellaneous Fecal Examinations (cont.) Fecal culture Used to differentiate parasites whose eggs or larvae are not easily distinguished by a fresh fecal sample Because the life cycle, pathogenicity, and epidemiology of some species differ, identification may be necessary for proper treatment and control May need an experienced helminthologist Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 24

25. Fecal Culture Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 25

26. Miscellaneous Fecal Examinations (cont.) Modified McMasters technique Provides an estimate of the number of eggs or oocysts per gram of feces Used mainly in livestock and horses Originally adapted from a technique used in people infected with hookworms Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 26

27. Modified McMasters Technique Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 27

28. Staining Procedures Stains may be used to recognize certain structural characteristics of trophozoites and cysts Lugol iodine and new methylene blue commonly used with direct smear Do not preserve the slide Help with identification If protozoal parasites cannot be identified on direct smears Fecal smears with suspected trophozoites can be dried and stained with Diff-Quik or Wright or Giemsa stain and sent to a diagnostic laboratory Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 28

29. Staining Procedures (cont.) Acid-fast staining is used to identify Cryptosporidium sp. Cryptosporidium is a parasite of the intestinal tract of many animals including humans Diff-Quik stain can be used to aid in identification of Isospora sp. Intestinal mucosal scrapings Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 29

30. Summary Methods to examine fecal specimens include gross and direct microscopic examinations Other examinations include fecal flotation, centrifugal flotations, and sedimentation Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved. 30