Co-Infection of cattle in Virginia withTheileria orientalis ikedagenotype andAnaplasma marginale

Abstract

Theileria orientalis ikeda is a newly identified agent of bovine infectious anemia in the United States. Although it is transmitted by separate tick hosts than Anaplasma marginale - a bacterial etiology of bovine infectious anemia -the geographic distributions of these two infectious organisms overlap, with co-infection reported in some cattle. Only anaplasmosis has approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex qPCR for A. marginale and T. orientalis. With a cut-off of 38 cycles, the duplex assay has a sensitivity of 96.97% and a specificity of 100% for A. marginale; with a cut-off of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, this qPCR facilitates the study of co-infection rate of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for the presence of T. orientalis, 125 were positive for the presence of A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. This indicated that co-infection of both of these etiologies of bovine infectious anemia does occur within the state of Virginia. It is likely that this pattern of infection will be seen in regions where T. orientalis and A. marginale are endemic, despite the difference in tick vectors.

Rapid Discovery and Detection of Haemaphysalis longicornis through the Use of Passive Surveillance and Collaboration: Building a State Tick-Surveillance Network

“…Additionally, verified specimens of H. longicornis were screened for animal-associated pathogens. Specifically, screening included Anaplasma and Ehrlichia via nested PCR amplification of the groEL genes [32], Babesia via nested PCR amplification of 18S rRNA [33], and Anaplasma and Theilieria via multiplex qPCR amplification of major surface protein 5 (msp5) and major piroplasm surface protein (mpsp), respectively [8]. Briefly, ticks were bisected longitudinally, and half of each specimen was saved as a voucher in a −20 • C freezer at UTK.…”

“…One positive control (previously positive tick with targeted DNA) and two negative controls (water and MasterMix without DNA, respectively) were used. For detection of A. marginale and T. orientalis Ikeda, we used a newly developed multiplex qPCR amplification of msp5 and mpsp [7,8]. If a tick was PCR positive (presence of band in a 1.5% agarose gel: 1 X TAE buffer with ethidium bromide for 2 h at 100 V), then that amplicon was bidirectionally sequenced at Eurofins Genomics (Louisville, KY, USA) using Sanger sequencing.…”

“…and Hepatozoon canis), and human pathogens (e.g., Borrelia, Ehrlichia, Rickettsia, and several viruses) [6]. Recently, investigators in Virginia determined field-collected H. longicornis were infected with the Theileria orientalis Ikeda genotype and demonstrated that study ticks were competent experimental vectors for the Ikeda strain of the pathogen in local cattle [7][8][9][10].…”