Developmental Studies of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in Male Dermacentor andersoni (Acari: Ixodidae) Infected as Adults by Using Nonradioactive in Situ Hybridization and Microscopy

Abstract

The development of Anaplasina marginale Theiler was studied in ticks using a nonradioactive in situ hybridization method developed in our laboratory. Male Rocky Mountain wood ticks, Dermacentor andersoni Stiles, were infected intrastadially by allowing them to feed for 7 d on an infected calf (acquisition feeding). The ticks were then removed and held in a humidity chamber for 5 d before being fed on a 2nd susceptible calf for 10 d (transmission feeding). Two groups of 10 ticks were collected daily during the 22-d experiment. In one group one-half of each tick was processed and embedded in paraffin and in the other group one-half of each tick was embedded in LR White for in situ hybridization. The companion tick halves from each group were fixed and embedded in Dow Epoxy Resin resin for routine light and electron microscopy. As detected by in situ hybridization on LR White- and paraffin-embedded sections and by microscopy, initial infection of A. marginale in ticks occurred in gut tissues either on the 7th d of acquisition feeding or the 1st d of the held period and infection persisted throughout transmission feeding. The highest number of ticks with gut infection was observed on the 5th d of transmission feeding. Salivary glands became infected with A. marginale on the 1st day of transmission feeding and remained infected throughout the transmission feeding period. Peak infection was observed on day 4 of transmission feeding. After the beginning of transmission feeding, A. marginale infection was also observed in interstitial, reproductive, skeletal muscle, fat body, and Malpighian tubule tissues. Although A. marginale infection of ticks clearly originates in midgut epithelial cells, many tissues eventually become infected during transmission feeding, resulting in a generalized infection. The infection of multiple tissues may contribute to the ability of A. marginale infection to persist in intrastadially infected male ticks.