Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation) showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

Bovine anaplasmosis or cattle-tick fever is a tick-borne haemolytic disease caused by the rickettsial haemoparasite Anaplasma marginale in tropical and subtropical areas of the world. While difficult to express, the proteins VirB9-1 and VirB10 are immunogenic components of the outer membrane type IV secretion system that have been identified as candidate antigens for vaccines targeting of A. marginale. Soluble VirB9-1 and VirB10 were successfully expressed using Pichia pastoris. When formulated with the self-adjuvanting silica vesicles, SV-100 (diameter: 50 nm, and pore entrance size: 6 nm), 200 µg of VirB9-1 and VirB10 were adsorbed per milligram of nanoparticle. The VirB9-1 and VirB10, SV-100 formulations were shown to induce higher antibody responses in mice compared to the QuilA formulations. Moreover, intracellular staining of selected cytokines demonstrated that both VirB9-1 and VirB10 formulations induced cell-mediated immune responses in mice. Importantly, the SV-100 VirB9-1 and VirB10 complexes were shown to specifically stimulate bovine T-cell linages derived from calves immunised with A. marginale outer membrane fractions, suggesting formulations will be useful for bovine immunisation and protection studies. Overall this study demonstrates the potential of self-adjuvanting silica vesicle formulations to address current deficiencies in vaccine delivery applications.

Animal health measures mainly rely on vaccination or chemical control for major pests and pathogens, causing issues of residue, toxicity and development of resistance. For example, control of Sheep flystrike and lice-infestation affecting the Australia’s sheep/wool industry (>3.5 B) have developed resistance to nearly all control chemicals used in the past. Topicals RNAi provides an innovative clean-green, non-toxic, environmentally sustainable biological control solution. Biodegradable clay particles as carriers can be used to deliver double stranded RNA (dsRNA), the key trigger molecule of RNA interference pathway. As an early proof of concept, we investigated the stability dsRNA loaded on two types of Clay particles: Clay 1 (releases dsRNA under acidic conditions) and Clay 2 (releases dsRNA under alkaline conditions) on cattle hide. Cattle skin was treated with Cy3 labelled dsRNA alone and Cy3 labelled dsRNA loaded on Clay1 or Clay2. The skin samples treated with the Cy3 formulations were imaged using confocal microscopy. Once imaged, the skin samples were washed and stored at room temperature for 5 days, later the samples were re-imaged to detect the fluorescent signal (Figure 1). The dsRNA loaded on clay particles was stable unlike naked Cy3-dsRNA which degraded and was not visible after washing. This increased inherent stability of the dsRNA molecules, combined with the environmental stability afforded by the Clay particles, offers promise to provide a sustainable solution for animal health. Topical RNAi can reduce reliance on trade withholding periods of meat/wool without chemical residues, enhance animal welfare and increase production of premium quality meat/wool, improve export potential, competitiveness and long-term profitability of livestock industry.