-167- 



suspended in 3% bacto-agar (Akin, pers. comm., 1984). They were 

 postfixed in 2% gl utaraldehyde in 0.2 M cacodylate buffer (pH 7.2) for 

 1-1/2 hrs. Sand flies that had fed on an infected hamster five and 

 six days previously were the source of promastigotes. The flies were 

 immobilized by refrigeration at -1°C for ten minutes and washed in 

 insect Ringer's solution (pH 7.2; Cavanaugh, 1956) to remove body 

 hairs. Whole flies or dissected guts were fixed in 2% gl utaraldehyde 

 in 0.2 M cacodylate buffer for 1-1/2 hrs before postfixation in 1% 

 osmium tetroxide for 1 hr. The fixed specimens were dehydrated in 

 graded concentrations of ethanol and cleared in absolute acetone. 

 They were infiltrated with graded concentrations of 30, 70 and 100% 

 Spurr's resin (Spurr, 1969) for 1, 1, and 12 hrs, respectively, 

 followed by imbedding in 100% Spurr's resin at 60°C for 48 hrs. 



Ultrathin sections were cut using an LKB Ultratome III, model 

 8800, and were poststained with uranyl acetate followed by lead 

 citrate. Specimens were subsequently examined using a Hitachi HI) 11-E 

 or Philips EM 301 electron microscope. 



Transmission Trials 



At first, potentially infected females were offered daily blood 

 meals on anesthetized, uninfected hamsters. It was found, however, 

 that they would not refeed prior to oviposition, so in all later 

 trials, second blood meals were offered only after the commencement of 

 oviposition. Potentially infected hamsters were checked at least 

 weekly for six months for signs of infection. Infections due to 

 Leishmania were confirmed by microscopic examination of Giemsa-stained 

 aspirates drawn from cutaneous lesions that subsequently developed. 



