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

Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K_D ∼ 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K_Ds of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.

The intracellular protozoal parasite Theileria orientalis has rapidly spread across South-eastern Australia, substantially impacting local cattle industries since 2006. Haemaphysalis longicornis appears to be a biological vector in the endemic regions. Mechanical transfer of blood by biting arthropods, in colostrum or iatrogenic transmission though husbandry procedures is another possible mode of transmission. This study assesses the risk of these mechanical modes of transmission. Blood was collected from a T. orientalis Ikeda positive Angus steer, and was 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 negative controls and the donor calf was also followed as a positive control. Blood was collected over 3 months, and analysed via qPCR for the presence of the parasite. Samples of the sucking louse Linognathus vituli were collected opportunistically from calves 5 months after inoculation and tested for T. orientalis. For the colostral transmission study, 30 samples of blood and colostrum were collected from cows at calving in an endemic herd. These samples along with blood from their calves were tested by qPCR for T. orientalis and for antibodies to the major piroplasm surface protein (MPSP). Eight of the nine inoculated calves became positive for T. orientalis. The prepatent period of these infections was inversely correlated with inoculation dose. All negative control calves remained negative and the positive control calf remained positive. Samples of L. vituli tested positive for T. orientalis Ikeda, while some samples of colostrum were also shown to be qPCR and anti-MPSP positive. All calves in the colostral study tested qPCR negative although one was antibody-positive. T. orientalis is capable of being mechanically transferred by intravenous inoculation with small volumes of blood and is detectable up to 5 months post-infection. 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 possible modes of disease transmission.

Chlamydia psittaci is an avian pathogen capable of spill-over infections to humans. A parrot C. psittaci strain was recently detected in an equine reproductive loss case associated with a subsequen...

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae- type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity.