Distribution of Developing and Mature Brugia malayi in Cats at Various Times after a Single Inoculation

Abstract

Young cats were infected with Brugia malayi by administering 50 to 105 infective larvae to puncture wounds on the volar surface of one rear foot. At various times from 3 days to 69 weeks after exposure to the infective larvae, the animals were killed and the number and location of developing and mature worms recorded. Soon after infection a large proportion of the larvae could be recovered in the popliteal node. As the infection progressed, a substantial number of larvae left the popliteal lymph node and migrated distally in the lymphatic vessels toward the foot where infection had been initiated. In no instance was any appreciable migration to other body sites, including other lymph nodes, seen. In cats killed 7 weeks or longer after infection, 36 to 84% of the infecting dose was recovered at necropsy. The significance of these findings as they pertain to drug efficacy studies is discussed. In earlier studies, attempts were made to develop techniques which would facilitate rapid and consistent recovery of Brugia malayi from experimentally infected animals. It was found that if cats were infected on the rear foot by using a puncture wound technique, a relatively large proportion of the larvae which were administered could be readily recovered soon after infection. From 1 to 10 days after infection, larvae were almost exclusively located in the popliteal lymph node and adjacent afferent lymphatic vessels (Ewert and ElBihari, 1971). To utilize these techniques efficiently, it is necessary to determine if the parasites remain in the area where they are first recovered, or if they migrate to other sites as the larvae develop and reach sexual maturity. This would be of particular importance in studies correlating pathology with the site of infection and in determining the effect of drugs on the parasite. MATERIALS AND METHODS Materials and methods were similar to those described earlier (Ewert and ElBihari, 1971). Aedes togoi were fed on cats with from 40 to 300 microfilariae of subperiodic Brugia malayi per 20 mm3 of blood. Two weeks after feeding, the mosquitoes were dissected in a drop of physiological saline to free the infective-stage larvae. Eightto 14-weekold cats, reared in mosquito-free quarters, were anesthetized by intraperitoneal injection of 25 to 30 mg/kg sodium pentobarbital. Hair on the volar Received for publication 23 February 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare. Grant No. AI 08260. surface of 1 rear foot was cut and 100 puncture wounds were made in the skin with a small needle. A 0.03-ml drop of physiological saline was placed over these puncture wounds. Infective Brugia malayi larvae, freed from dissected mosquitoes, were immediately transferred individually to the drop of saline over the puncture wounds by using a small needle mounted on an applicator stick. Animals were not moved until the drop of saline had completely disappeared, approximately 30 min. At various times after exposure to infective larvae, cats were killed with an overdose of sodium pentobarbital. To demonstrate lymph nodes and vessels, 0.1 to 0.5 ml of a 0.4% aqueous solution of direct Sky Blue dye (Wyeth Laboratories) was injected into the foot pads, side of head, and base of tail, 15 to 60 min prior to killing the animals. Lymph nodes and superficial lymphatic vessels were exposed by reflection of the skin in the appropriate areas. After examining exposed nodes and lymphatic vessels with a stereoscopic :microscope, the nodes and as much of the vessels as possible were removed intact and placed in saline. Popliteal, inguinal, axillary, and cervical nodes along with their afferent and efferent vessels were teased with dissecting needles and examined for parasites. The intact living and dead parasites were removed and counted. A squash preparation between glass slides was then made of the nodes and lymphatic vessels and examined at higher magnification to detect any parasites missed earlier. Additionally, the rear legs and skin of rear quarters of all animals and the entire carcass and skin of several selected animals were soaked in warm saline for several hours. The saline was then decanted and the sediment examined for parasites.