FILARIASIS RESEARCH REAGENT RESOURCE CENTER:
University of Wisconsin Oshkosh
STANDARD OPERATING PROCEDURE
TITLE: Experimental Direct Infection of Gerbils and Hamsters with Acanthocheilonema viteae ThirdStage Larvae (L3)
SOP NUMBER: A. vit. 8
1. PURPOSE
This standard operating procedure outlines the experimental infection of gerbils and hamsters with
Acanthocheilonema viteae by injection of infective third-stage larvae (L3).
2. MATERIALS
2.1. Acanthocheilonema viteae L3 in medium of choice (i.e., HBSS or RPMI 1640 supplemented with
penicillin and streptomycin)
2.2. Anesthesia of choice (i.e., isoflurane)
2.3. Source of heat to provide thermal support for the animal during anesthesia
2.4. 70% ethanol
2.5. Small hair clippers
2.6. Surgical skin glue
2.7. One inch 20 gauge needle
2.8. 1 cc syringe
2.9. Micropipette
3. PROCEDURES
3.1. Collect A. viteae L3 per SOP Number A. vit. 6: Collection of Infective Larvae of
Acanthocheilonema viteae from Infected Ticks (Ornithodoros tartakowskyi) and transfer the
desired number of L3 to a 1 cc syringe using a micropipette.
Note: When injecting larvae into gerbils or hamsters, the solution must be quite clean (i.e., free
of debris and not "cloudy"), to avoid infection and possible death of the vertebrate hosts.
Last revision: 3/5/14 S.S.
A. vit. 8
Note: Acanthocheilonema viteae infection in gerbils has been reported to cause neuropathology,
but the incidence of pathology is low in infections using small numbers of the parasite. The
recommended number of L3 per gerbil infection is 10-15, and the recommended number of L3
per hamster infection is 300-400. These numbers can be adjusted per your IACUC approval.
When injecting hundreds of L3, it is recommended to split the worms into multiple injections.
This can prevent a ball of worms forming that is too large to pass through the needle during
injection.
3.2. If desired, anesthetize the animal with isoflurane or other anesthesia of choice (This procedure
can be performed on a manually restrained animal or on an anesthetized animal according to an
approved IACUC protocol.).
For gerbils: Use 5% isoflurane to induce the animal, use 2.5-3.5% isoflurane for maintenance
anesthesia during the procedure. Isoflurane is used as a mixture with oxygen (100%) at a flow
rate of 1 L/min.
For hamsters: Use 4% isoflurane to induce the animal, use 2.5-3.0% isoflurane for maintenance
anesthesia during the procedure. Isoflurane is used as a mixture with oxygen (100%) at a flow
rate of 1 L/min.
3.3. Position the animal in ventral recumbency on the source of thermal support.
Note: Prior to ever using the heat source you choose, be sure to test it to ensure it does not get too
hot, which could result in death of the animal or serious burns. A stockinet sleeve filled with
uncooked rice warmed briefly in the microwave (i.e., 30-45 seconds) works well, as it holds heat
well and allows for proper positioning of the animal. The rice filled stockinet can also be
autoclaved between uses.
3.4. Prepare the animal by clipping the hair off of the animals back, directly dorsal to the scapulae.
Swab the skin at that location with 70% ethanol and a sterile cotton tipped applicator.
3.5. A 1 inch 20 gauge needle is attached to the 3 cc syringe and the contents of the syringe are
injected subcutaneously into the scapular region. Ideal injection volume is 0.5-0.8 cc. When
large volumes of media are injected subcutaneously, a greater percentage of the medium will
leak out of the injection site.
After the initial injection, the needle is left in place in the subcutaneous layer of the animal, and
the syringe is removed. Sterile medium is added to the syringe, the syringe is reattached to the
needle, and the medium is injected through the needle to ensure all L3 are flushed from the
syringe; this step may be repeated several times up to a total volume of 3 cc.
3.6. Upon removal of the needle, use two fingers to tent the skin at the injection site and provide light
pressure for ~15 seconds to help prevent leakage. The small hole in the skin can be sealed with a
small bead of surgical skin glue to prevent leakage.
3.7. Both gerbils and hamsters are expected to be microfilaremic 50-60 days post infection.
3.8. Time to Completion: Once L3s are collected, infection of vertebrate hosts takes approximately
15 minutes per animal
Last revision: 3/5/14 S.S.
A. vit. 8
Last revision: 3/5/14 S.S.
A. vit. 8