Background Theileriaare 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 project provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite. This includes identifying the likely biological vector tick, as well as assessing the role of biting arthropods (biting flies, lice, mosquitos and ticks) and blood transmission during management procedures in the transmission of the disease. Transplacental and colostral transfer were also investigated. Methods To identify possible vectors for disease, ticks, biting flies, lice and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife. 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 DNA and the genotypes Ikeda, Chitose and Buffeli using real-time PCR. To further assess mechanical transmission and iatrogenic transmission through husbandry procedures, blood was collected from a T. orientalis Ikeda positive Angus steer, and cryopreserved in liquid nitrogen. Blood was also collected from a T. orientalis Ikeda positive Holstein-Friesian calf, but was not cryopreserved. Blood was then inoculated inoculated into the jugular vein of 9 calves in 3 treatment groups, each with 3 animals. Calves in group 1 received 10 ml of cryopreserved blood, while those in groups 2 and 3 received 1 ml (fresh blood) and 0.1 ml (cryopreserved), respectively. An additional three animals remained as non-inoculated (negative) controls and the donor calf was also followed as a positive control. Blood was collected at intervals for 3 months, and analysed via qPCR for the presence of the parasite. Thirty samples of colostrum together with blood samples from the respective neonatal calves at 3-6 weeks along with their dams, from a commercial dairy herd within an endemic region in Gippsland, were collected and also tested for T. orientalis antigen and antibodies to the major piroplasm surface protein (MPSP). To investigate transplacental transmission, 30 cows and their calves had blood collected within 24 hours of the cow giving birth. These EDTA samples were then tested via qPCR for T. orientalis. 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, andCulex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquito pools. The haematophagousflies tested negative. The batches of sucking louse (Linognathus vituli) from infected calves tested positive for T. orientalis Ikeda. In the passive transfer trial, eight of the nine calves became positive for T. orientalis. Those calves given a high dose (10 ml) became positive 28 days following transfusion. Calves transfused with 1 ml became positive 41 days following transfusion, while 2 calves in group 3 became positive for the Ikeda genotype at 66 days, and the third animal became positive 98 days following transfusion. All negative control calves remained negative and the positive control calf remained positive. Samples of colostrum were also shown to carry T. orientalis. Blood samples from calves receiving this colostrum were tested between the ages of 3-6 weeks and showed no transmission. However, the transfer of antibodies from colostrum was seen. Transplacental transmission was not observed in this study. Conclusions This is the first demonstration of a potential vector (Haemaphysalis longicornis) for T. orientalis in the current Australasian disease outbreak. T. orientalis is capable of being transferred mechanically by intravenous inoculation with small volumes of blood. However, infection by this route does not appear to result in a high level of infection (as determined by qPCR) and did not result in any overt clinical signs. Animals infected by this means may play a significant role in the transmission of the disease by acting as asymptomatic carriers. Other modes of blood transfer, including biting arthropods and colostral transfer are also likely to play a role in transmission, and might help explain the rapid spread of the disease in Australasia. Transplacental transmission seems unlikely to occur.