Theileria cervi and Babesia odocoilei Infections Detected in Deer Hosts
by Specific SSU rRNA Gene Fragment Amplification
 
P. J. Holman, J. Bennett, D. Cruz and G.G. Wagner
Department of Veterinary Pathobiology,
Texas A&M University, College Station, Texas, USA

Blood samples from white-tailed deer, elk and caribou with hemoparasitic infections were tested by amplification of the SSU rRNA gene using primers designed for specific amplification of Theileria cervi or Babesia odocoilei gene fragments. Theileria and Babesia isolates were acquired over a ten-year period from animals with clinical histories including (1) mortalities due to babesiosis, (2) high T. cervi parasitemia and associated anemia and (3) apparently normal animals with no clinical signs of infection. Samples included Theileria cervi from elk in Canada, Wisconsin and Oklahoma and from white-tailed deer in Oklahoma, and Babesia odocoilei from white-tailed deer in Texas, elk in Wisconsin and a caribou in Minnesota. Giemsa stained blood films from each sample were examined microscopically. 

Genomic DNA was purified from blood samples or cultured parasites using a standard phenol-chloroform extraction method. A two-step amplification protocol was developed. In the first step, universal primers for amplification of eukaryotic SSU rRNA genes were used under conditions specific for the amplification of the parasite gene from the purified genomic DNA. In the second step, T. cervi or B. odocoilei SSU rRNA gene fragments were amplified from the SSU rRNA gene amplicon obtained in the first step using specific primers for each organism. Amplification products were separated by electrophoresis through 1% agarose gels, stained with ethidium bromide and viewed by UV transillumination. 

Theileria cervi and B. odocoilei specific amplicons were obtained from all blood samples positive by Giemsa-stained blood film examination for the respective parasite. In addition, a dual infection of T. cervi and B. odocoilei was detected in an elk known to be infected with the latter parasite. A specific B. odocoilei amplicon was not obtained from a California caribou that was observed to be positive by culture and Giemsa-stained blood film examination. SSU rRNA gene sequence analysis confirmed that the parasite present was distinct from B. odocoilei.