Protocols for field and laboratory rodent studies - HAL

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Definitions4- Isolating helminths in rodents4. Isolation of helminths from rodents(Alexis Ribas Salvador, Kittipong Chaisiri, Serge Morand, Jean-Pierre Hugot, Voitto Haukisalmi and HeikkiHenttonen)Helminths, also called parasitic worms, constitute a division of eukaryotic macroparasites that liveinside their host. They can be found in different locations, known as microhabitats (body cavity, lungs,stomach, small intestine, caecum, blood…). They derive nourishment by disrupting their hosts' nutrientabsorption and circulation, sometimes causing disease.Rodents are definitive or intermediate hosts for adult or larval stages of helminths, respectively.Helminths are categorized into four groups (Cestoda, Nematoda, Trematoda and Acanthocephalans).Pentastomids are also sometimes studied by helminthologists, as they are recovered duringdissections. In rodents, helminth size varies in length from few millimeters to tens of centimeters (thelongest are found within the Cestoda).Cestoda (tapeworms)- Segmented plane, covered with tegument.- Presence of a scolex with 4 suckers and sometimeswith an armed rostrellum in adults.- Hermaphrodites.Nematoda (roundworms)- Cylindrical shape, covered with collagenous cuticle.- Various styles of head, with / without lips and teeth.- Dioecious.Figure 56: Scolex of Raillietina sp. with 4 suckers andarmed rostellum (20x) (Photo: Ribas A. and Chaisiri K.)Trematoda (flukes)- Unsegmented plane, covered with tegument.- Presence of two suckers (oral and ventral orposterior sucker), rarely one.- Hermaphrodites.Figure 57: Filarid worm(Photo: Ribas A. and Chaisiri K.)Acanthocephala (thorny-headed worms)- Externally segmented and round, covered with finecuticle.- Presence of a protrusible proboscis, covered withhooks, to pierce the gut of its host. No mouth.- Dioecious.Figure 58: Notocotylus loeiensis(Photo: Ribas A. and Chaisiri K.)Figure 59: Moniliformis moniliformis with pseudosegmentedbody (Photo: Ribas A. and Chaisiri K.)4.1. Gastrointestinal (GI) tract and helminths isolationRecommendations:Helminths should be separated by organs,cestodes relaxed in water, and preserved in70% ethanol in a tube. Tubes should becarefully labeled with the animalidentification number and name of organ.Do not use formalin to store specimens thatwould prevent genetic analysis.Isolate the gastrointestinal tract, as soon aspossible.Figure 60: GI dissection in Kalasin province, Thailand(Photo: Herbreteau V.)32
4- Isolating helminths in rodentsProtocol:1- During the dissection, check each organ for thepresence of helminths. Especially check:• Liver where Taenia and other tapeworm cystsmay be present in some species (Fig. 61A).Cysts should be separated and preserved inethanol (Fig. 61B). If a cyst is opened, slowlyand carefully pull the worm out of the cyst,avoiding any damage. Larval tapeworm cystscan also occur freely in body cavities• Lungs. Although lungs are collected to screenfor other parasites and pathogens, theyshould be observed for the eventual presenceof Angiostrongylus sp.• Thoracic cavity, where Filaridae can be found(Fig. 62).Figure 61: Cysts of cestode (Taenia taeniaeformis)isolated from liver (Photo: Herbreteau V.)2- Separate the gastrointestinal tract: cut the endof rectum before the anus and lift the GI tract(Fig. 63). Carefully cut ligaments connecting theGI tract to the dorsal side of the body cavity.Separate it from the liver and finally cut it from theesophageal sphincter of stomach.3- If the gastrointestinal tract cannot beexamined the same day,- but within one or two days: GI tract can be keptin a refrigerator and the material remains goodenough for microscopical and genetic studies. GItract should be placed in a small zipper bag, airpressed out.- or not in a few days: place the GI in a zipperbag, press air out and freeze (normal freezer isOK). Frozen and later thawed helminth material isperfect for microscopical and genetic work.- otherwise (this is the least preferred alternative):place the GI tract in a hermetic plastic box,Falcon tube (Fig. 64 and 65) or even a plasticbag, filled with 80% ethanol as a fixative agent. Ifpossible, ethanol should be changed the next daybecause released water from GI tissue will dilutethe ethanol. Cestode material fixed this way iscontracted, not relaxed, and can seldom be usedfor detailed microscopic morphology (speciesdescriptions).Figure 62: Filaria from the lung cavity(Photo: Herbreteau V.)Figure 63: Separation of the gastrointestinal tract(Photo: Herbreteau P.)For a better conservation, aminimum of 4 volumes ofethanol should be added toone volume of viscera. Do notforget to put an identificationtag with the GI. As tags tendto be attached to viscera andbecome dirty with blood, useplastic tags (such as Dymo ® )instead of normal paper. Besure the container iscompletely hermetic to avoidevaporation as dry visceraare not suitable forhelminthological study. Incase containers used in thefield are not big enough,renew ethanol once back inthe laboratory; this will allow abetter conservation ofhelminths.Figure 64: Preservation of GI inFalcon tubes (Photo: Chaisiri K.)Figure 65: Preservation of Falcon tubesin a container filled with 70% ethanol toavoid evaporation (Photo: Herbreteau V.)33
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Protocols for field and laboratory rodent studies - HAL

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