DETECTION OF ANAPLASMA-MARGINALE (RICKETTSIALES, ANAPLASMATACEAE) IN HEMOLYMPH OF DERMACENTOR-ANDERSONI (ACARI, IXODIDAE) WITH THE POLYMERASE CHAIN-REACTION

The polymerase chain reaction (PCR) was used to detect Anaplasma margi nale in hemolymph collected from live Dermacentor andersoni Stiles tic ks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment wa s found to be the optimum method of hemolymph preparation for PCR. Hem olymph samples were collected and pooled from adult ticks exposed as n ymphs on days 0-10 of feeding on a susceptible calf. For male and fema le ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the pa rasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAG CAG CAGCAAGACCT-FCA-3'), which flank a 409-bp fragment of the A. margi nale Florida isolate msp1beta gene. Infected tick hemolymph was PCR-po sitive for A. marginale at all collection times, including unfed adult s infected as nymphs and previously unexposed adults that fed on infec ted calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potenti ally without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for fur ther studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A . marginale isolates from diverse geographical areas of the United Sta tes.