Helminth Collection and Identification from Wildlife
Summary
Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are “roundworms” (nematodes), “thorny-headed worms” (acanthocephalans), “flukes” (trematodes), and “tapeworms” (cestodes). Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified.
Abstract
Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are "roundworms" (nematodes), "thorny-headed worms" (acanthocephalans), "flukes" (trematodes), and "tapeworms" (cestodes). The optimum method for collecting helminths is to examine a host that has been dead less than 4-6 hr since most helminths will still be alive. A thorough necropsy should be conducted and all major organs examined. Organs are washed over a 106 μm sieve under running water and contents examined under a stereo microscope. All helminths are counted and a representative number are fixed (either in 70% ethanol, 10% buffered formalin, or alcohol-formalin-acetic acid). For species identification, helminths are either cleared in lactophenol (nematodes and small acanthocephalans) or stained (trematodes, cestodes, and large acanthocephalans) using Harris' hematoxylin or Semichon's carmine. Helminths are keyed to species by examining different structures (e.g. male spicules in nematodes or the rostellum in cestodes). The protocols outlined here can be applied to any vertebrate animal. They require some expertise on recognizing the different organs and being able to differentiate helminths from other tissue debris or gut contents. Collection, preservation, and staining are straightforward techniques that require minimal equipment and reagents. Taxonomic identification, especially to species, can be very time consuming and might require the submission of specimens to an expert or DNA analysis.
Introduction
Vertebrates are parasitized by four major groups of helminths (worms). Two of the groups, trematodes, or flukes, and cestodes, or tapeworms, fall within the Phylum Platyhelminthes. The other two groups are the nematodes, or roundworms, (Nematoda) and the acanthocephalans, or thorny-headed worms (Acanthocephala). Many of these parasites have been documented as causes of morbidity and mortality in wild birds and mammals1,2.
Most helminths have complex life cycles involving more than one host1,2 . For instance, trematodes have one or two intermediate hosts (usually invertebrates) and a final host. All hosts need to be available for the cycle to be completed and thus for adult helminths to be present in the final hosts (which are the topic of this manuscript). So it is important to keep in mind that seasonal fluctuations can occur in the prevalence and intensity of some helminth species and long-term monitoring is desirable to capture the complete helminth of auna of a particular host.
The optimum method for collecting helminths is to examine a host that has been euthanized. This allows for the collection of helminths while they are still alive and for their proper relaxation and fixation. Material from hosts that have been dead more than 24 hr, or have been frozen and thawed for examination, is often inferior and difficult to identify. Trematodes and cestodes from frozen or formalinized hosts are often badly contracted and have lost structures crucial to identification like oral spines on trematodes or the hooks on the scolex of tapeworms. However, the reality is that most material available from vertebrate hosts these days is frozen or preserved.
Databases containing DNA sequence information for helminths is quickly growing and thus taxonomic identification is already possible for many species. Therefore, helminths should be preserved for potential DNA analyses as much as possible. However, DNA of good quality is not possible if specimens are fixed in formalin. Methods outlined here for collecting and killing helminths will yield material that is suitable for DNA extraction and genetic analyses.
Below, we describe detailed methods on how to necropsy vertebrates (amphibians, reptiles, birds and mammals; monogenean trematodes from fish are not included) for the collection of helminths, followed by procedures on how to preserve and process them for taxonomic identification.
Protocol
Representative Results
Discussion
It is extremely difficult to identify helminth parasites, even to genus, based on poor material. Cestodes and trematodes, in particular, tend to die and deteriorate fairly rapidly after the death of the host. The taxonomy of cestodes depends greatly on the number, size, and shape of rostellar hooks, which are often lost in frozen material. The same applies to certain trematodes with spines around the oral sucker such as echinostomes and heterophyids, which are also frequently lost. Because of their thick cuticles, nemato…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Dr. Joe N. Caudell, Disease Biologist for the Indiana USDA APHIS Wildlife Services Program at Purdue University, for providing specimens used for the collection of helminths for the production of this video. Jennifer Serafin prepared all of the chemical solutions.
Materials
| Fixing | |||
| Alcohol-Formalin-Acetic Acid (AFA) | Fisher Scientific | A407-1 (ethanol) F75F-1GAL (formaldehyde) BP2401500 (glacial acetic acid) |
Ethanol 85% (85 ml) + formaldehyde 37% (10 ml) + glacial acetic acid (5 ml) |
| Ethanol for killing and long-term preservation | Fisher Scientific | A407-1 | Ethanol 100% (70 ml) + distilled water (30 ml) |
| Ethanol for fixing and DNA studies | Fisher Scientific | AC61511-0010 | Ethanol 80-90% (80-90 ml) + distilled water (20-10 ml), respectively |
| Formalin Buffered | Fisher Scientific | SF100-4 | Formalin 10% (10 ml) + distilled water (90 ml) |
| Glycerine-alcohol | Fisher Scientific | A407-1 (ethanol) AC15365-1000 (glycerine) |
Ethanol 70% (95 ml) + glycerine (5 ml) |
| Clearing, staining and mounting | |||
| Ethanol for dehydrating during staining | Fisher Scientific | A407-1 | Ethanol 80%, 95%, 100% (80 ml, 95 ml, 100 ml) + distilled water (20 ml, 5 ml, 0 ml) |
| Lactophenol | Fisher Scientific | R400-27 | – |
| Phenol | Fisher Scientific | A931I-1 (phenol) A407-1 (ethanol) |
Phenol 100% (80 ml) + ethanol 100% (20 ml) |
| Harris’ Hematoxylin | Fisher Scientific | S25347 (hematoxylin) A407-1 (ethanol) A567-500 (ammonium aluminum sulfate) S25553 (sodium iodate) |
Hematoxylin (5 g) + ethanol 100% (50 ml) + ammonium aluminum sulfate (100 g) + sodium iodate (0.37 g) + distilled water (1,000 ml) |
| Semichon’s acetic carmine | Fisher Scientific | BP2401500 (glacial acetic acid) S25236 (carmine) A407-1 (ethanol) |
Glacial acetic acid (100 ml) + carmine (1.5 g) + distilled water (100 ml). Heat in a boiling water bath for 15 min and cool. Filter to make stock solution. Mix 1:2 with 70% alcohol for staining. |
| Xylene | Fisher Scientific | X4-4 | Use only under a chemical fume hood. |
| Methyl salicylate | Fisher Scientific | S25437 | – |
| Ethanol basic | Fisher Scientific | A407-1 (ethanol) S25159A (concentrated ammonium hydroxide) |
Ethanol 70% (100 ml) + ammonium hydroxide (0.1 ml) |
| Ethanol acid | Fisher Scientific | A407-1 (ethanol) SA9233-100 (hydrochloric acid) |
Ethanol 70% (99.5 ml) + hydrochloric acid (0.5 ml) |
| Canada balsam | Fisher Scientific | B10-100 | – |
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