TITLE: Experimental Infection of Dogs, Cats,
Ferrets, and Rodents with Dirofilaria immitis,
Brugia malayi, B.
pahangi, or Dipetalonema reconditum
SOP NUMBER: 8.5
REVISION NUMBER: 2
1.0 PURPOSE
This
standard operating procedure outlines the
experimental infection of dogs, cats, ferrets, and
rodents with D. immitis, B. malayi, B.
pahangi, or D. reconditum by inoculation
of infective third-stage larvae (L3).
2.0 PROCEDURES
2.1 Collection of the L3
2.1.1 A stereo dissecting scope and a
micropipette (i.e., modified pasteur pipette) are
used to remove the infective larvae (L3)
from the petri dishes.
2.1.2 The L3 are counted into separate
petri dishes for each individual animal. The number
of L3/animal will vary according to
species of parasite and animal and the purpose of
the study.
2.1.3 After counting is completed, the petri dish
containing the L3 is tilted slightly to
allow the L3 to settle to the lowest part
of the dish.
2.1.4 Excess fluid is removed from the dish using
a pasteur pipette, such that less than 1 ml of fluid
(containing L3) remains in the dish.
2.1.5 The fluid containing the L3 is
pulled up into a 1-cc tuberculin syringe.
2.2 Subcutaneous injection of L3
(≤
400 L3 / animal)
2.2.1 A 20-gauge, 1.5-inch needle is
attached to the syringe and the contents of the
syringe are injected subcutaneously in the
inguinal area of the animal. After the initial
injection, media is added to the syringe and the
solution is injected to ensure that all L3
are removed from the syringe; this step may be
repeated several times. After the last
injection, media is added to the syringe and the
contents are emptied into a petri dish and
examined for remaining L3. For B.
pahangi and B. malayi, the L3
may be injected SC in the dorsum of one or more
of the paws of dogs and cats.
2.3 Intraperitoneal injection of L3
(≤ 1,000
L3 / animal)
2.3.1 For infecting rodents intraperitoneally
with B. malayi or B. pahangi, inject
larvae into the peritoneal cavity as described in
2.2.1. A 1.0- or 1.5-inch needle may be used.
3.0 Inoculum size by animal species, filarial
species, and infection route.
3.1 Inoculum size and route of infection may
vary by study protocol objective and requirements.
3.2 Guidelines for establishing infections
3.2.1 Dirofilaria immitis
3.2.1.1 Dogs (~35
L3 SC per dog to produce microfilaremic
infections; up to 400 L3 SC per dog for
donor dogs).
3.2.1.2 Cats (~100
L3 SC per cat to produce transient
microfilaremic infections).
3.2.1.3 Ferrets (~15
L3 SC per ferret to produce transient
microfilaremic infections; up to 75 L3 SC
per ferret for donor ferrets).
3.2.2 Dipetalonema reconditum
3.2.2.1 Dogs (up to 200 L3 SC to produce
microfilaremic infections).
3.2.3 Brugia pahangi
3.2.3.1 Dogs and cats (up to 400 L3 SC
per animal to produce microfilaremic lymphatic
infections).
3.2.3.2 Jirds and other rodents (up to 150 L3
SC to produce microfilaremic lymphatic infections; ~400 L3
SC to produce intraperitoneal infections,
particularly in the male jirds).