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.