Detection of Theileria orientalis genotypes in Haemaphysalis longicornis ticks from southern Australia

Abstract

Background: Theileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries. This paper provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite.

Methods: To identify possible vectors for disease, ticks, biting flies and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife, and mosquitoes and biting flies were collected in traps in close proximity to outbreak herds. Ticks were identified via DNA barcoding of the mitochondrial cytochrome oxidase I gene. Barcoded ticks were pooled according to species or phylogenetic group and tested for the presence of T. orientalis and the genotypes Ikeda, Chitose and Buffeli using real-time PCR.

Results: DNA barcoding and phylogenetic analysis identified ticks from the following species: Haemaphysalis longicornis, Ixodes holocyclus, Ixodes cornuatus, Ixodes hirsti, and Bothriocroton concolor. Additional Haemaphysalis, Ixodes and Bothriocroton spp. were also identified. Of the ticks investigated, only H. longicornis ticks from cattle carried theilerial DNA, with the genotypes Ikeda, Chitose and Buffeli represented. Mosquitoes collected in close proximity to outbreak herds included; Aedes camptorhynchus, Aedes notoscriptus, Coquillettidia linealis, Culex australicus, and Culex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquitoes. The haematophagous flies tested negative.

Conclusions: This is the first demonstration of a potential vector for T. orientalis in the current Australasian disease outbreak.

Figure 1

Distribution of Haemaphysalis longicornis in Australia. Map of Australia showing the known range of H. longicornis as described in [30]. An enlarged map of the state of Victoria is also shown with the geographic locations, from which ticks and other arthropods were collected, highlighted (closed circles). The location of the likely entry point of T. orientalis Ikeda into the state of Victoria is also shown (open circle).

Figure 2

Phylogenetic tree of ticks investigated. Phylogenetic tree based on mitochondrial cytochrome oxidase I (COI) gene sequences of ticks collected in this study along with reference tick COI sequences sourced from Genbank. The phylogenetic tree is based on a MUSCLE alignment of the sequences in which gap positions were trimmed and was constructed using the Maximum Likelihood method based on the model of Tamura and Nei, within the program MEGA v6 [41]. The tree with the highest log likelihood is shown. Bootstrap replications (1000) were performed, which are expressed on each node as a percentage. The scale bar indicates the number of nucleotide substitutions per site. Accession numbers for references sequences are indicated. Sequences derived from this study were deposited in GenBank (Accession numbers - to be provided). Species (or clades) identified in this study are indicated on the right.