الديدان الطفيلية جمع وتحديد الهوية من الحياة البرية
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
ومطفول الحيوانات البرية عادة من قبل مجموعة واسعة من الديدان الطفيلية. أربعة أنواع رئيسية من الديدان الطفيلية هي "الديدان" (النيماتودا)، "الديدان شوكية الرأس" (acanthocephalans)، "المثقوبة" (الديدان المثقوبة)، و "الدودة الشريطية" (الديدان الشريطية). الأسلوب الأمثل لجمع الديدان هو دراسة المضيف الذي كان القتلى أقل من 4-6 ساعة لأن معظم الديدان سوف يكون لا يزال على قيد الحياة. ينبغي إجراء التشريح شامل وجميع الأجهزة الرئيسية فحصها. يتم غسلها الأجهزة أكثر من 106 ميكرون غربال تحت الماء ومحتويات فحصها تحت المجهر ستيريو تشغيل. تحسب جميع الديدان ويتم إصلاحها عدد ممثل (إما في الايثانول 70٪، 10٪ من الفورمالين مخزنة، أو حمض الخليك، الكحول الفورمالين). لتحديد الأنواع، يتم مسح إما الديدان الطفيلية في lactophenol (الديدان الخيطية وacanthocephalans الصغيرة) أو الملون (الديدان المثقوبة، الديدان الشريطية، وacanthocephalans كبيرة) باستخدام الهيماتوكسيلين هاريس أو اللون القرمزي Semichon ل. الديدان هي KEYEد على الأنواع من خلال دراسة الهياكل المختلفة (على سبيل المثال. الشويكات الذكور في الديدان الخيطية أو منقاف في الديدان الشريطية). البروتوكولات المذكورة هنا يمكن تطبيقها على أي حيوان فقاري. أنها تتطلب بعض الخبرة في الاعتراف الأجهزة المختلفة والقدرة على التفريق الديدان الطفيلية من غيرها من الحطام الأنسجة أو الأمعاء محتوياتها. جمع وحفظ وتلطيخ هي التقنيات التي تتطلب واضحة الحد الأدنى من المعدات والكواشف. تحديد التصنيفية، خصوصا أن الأنواع، يمكن أن يكون وقتا طويلا جدا، وربما يتطلب تقديم العينات إلى خبير أو تحليل الحمض النووي.
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…
Disclosures
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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