Collecting Infective Larvae of Brugia pahangi, B. malayi, Dirofilaria immitis, and Dirofilaria repens From Infected Mosquitoes

Printer Version: Collecting Infective Larvae

TITLE:  Collecting Infective Larvae of Brugia pahangi, B. malayi, Dirofilaria immitis, and Dirofilaria repens From Infected Mosquitoes

SOP NUMBER:  8.4

REVISION NUMBER:  3

 

1.0       PURPOSE

This documents the procedures used in collecting infective larvae of Brugia pahangi, B. malayi, Dirofilaria immitis, Dirofilaria repens, and other mosquito-borne filariae from infected mosquitoes.

 

2.0       PROCEDURES

  • 2.1        Remove sugar cubes and water-soaked cotton from the cartons used to raise mosquitoes as described in SOP 8.2.
  • 2.2        Remove dead mosquitoes by aspiration from the carton (this may be performed as early as 24 to 48 hours prior to procedure 2.3).
  • 2.3        Place cotton containing ether on the nylon screen and then place the carton in a plastic bag for several minutes.  This procedure must be done under an explosion-proof hood.  Alternatively, the mosquito cartons may be placed in a freezer for approximately one minute.
  • 2.4        After all mosquitoes are immobilized, remove the top of the carton.  The infected mosquitoes (maximum of 2,250 mosquitoes) may be transferred to a piece of glossy paper (or similar smooth material).  Replace the screened lid of the carton in case infected mosquitoes remain in the carton.  If the infected mosquitoes are not transferred to a piece of glossy paper (or equivalent), proceed to procedure 2.5 and replace the screened lid of the carton.
  • 2.5        Quickly put the infected mosquitoes into a mortar without solution and gently crush them.  Add 2 to 3 ml of chilled (refrigerated) Hanks’ balanced salt solution (HBSS, pH 7.0) containing pen-strep (final concentration= 0.4 units penicillin/ml, 0.4 mcg streptomycin/ml, PS) to the mortar and  gently crush the mosquitoes again.
  • 2.6        The crushed mosquitoes on the pestle and in the mortar are then rinsed with the chilled HBSS-PS onto a 150 mesh sieve contained in a plastic petri dish.
  • 2.7        The sieve containing the crushed mosquitoes is gently, but quickly, agitated to removed scales, eggs, and debris body parts (e.g., legs) of the mosquitoes.  This procedure is repeated 3 to 4 times, using several dishes of fresh, chilled HBSS-PS.  As approximately 10% of the larvae may remain in these washings, it is advisable to keep these dishes until one has collected a sufficient number of larvae, i.e., it may be necessary to use all of the larvae from the washings to obtain a sufficient number of larvae.
  • 2.8        The sieve is then removed to fresh, warm (microwave on high level for a minimum of 90 seconds and a maximum of 105 seconds) HBSS-PS to allow the larvae to migrate out of the mosquitoes; the sieve is transferred to petri dishes containing fresh HBSS-PS every 15 to 30 minutes.  (Note: most of the Brugia  larvae will have migrated out of the mosquitoes within about 2 hours.  Larvae of D. immitis are less active, therefore, it is necessary to allow 1 to 2 more hours for collecting these larvae.)
  • 2.9        When inoculating Brugia larvae intraperitoneally into jirds, the solution must be quite clean, i.e., free of debris and not “cloudy,” to avoid peritonitis and possibly death of the jirds.  Thus, the solution should be relatively “clean” before inoculation of larvae either SC or IP, but particularly before IP injections..