Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Modified Atmosphere Packaging (MAP) for Control of Black Spot Formation in Chilled Prawns

The marketing of organically labeled prawns is predominately in a cooked or raw frozen form to avoid the development of melanosis (black spot). Certification for organic status prohibits the use of any added chemicals. The application of 60% CO2/40%N2 modified atmosphere to chilled (raw) prawns using two species of prawn was investigated for the ability to control black spot formation. Sensory assessment and microbiological counts were used to determine the end of product shelf life. Modified atmosphere packaged (MAP) prawns exhibited no melanosis for up to 16 days. The high quality life was retained for 12 days; shelf life of 16 days, according to standard microbiological criteria, was achieved, which is more than twice previously reported for non-MAP prawns. Results suggest MAP may be an effective method for the marketing of organically grown prawns as well as those produced by conventional prawn aquaculture without application of the normal chemicals used to prevent black spot. Copyright © 2014 Crown Copyright.

Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health.

Modified atmosphere packaging effects on the postharvest quality of papaya fruit

Export of Fijian papaya (Carica papaya) fruit to destinations such as New Zealand has increased significantly over the last several years. Shipment by sea rather than air is the preferred method, given the capacity for larger volumes and reductions in cost. Long shipping times, however, can compromise fruit quality, although the use of modified atmosphere packaging (MAP) may provide a viable solution for extending fruit storage life. In a collaborative ACIAR project, Australian and Fijian researchers investigated the potential of using MAP to extend storage life of a Fijian papaya ('Fiji Red') fruit based on simulated sea transport conditions. Fruit were packed in one of three MAP environments within cartons, consisting of either a (1) Low Density Polyethylene (LDPE) bag with 10 g of KMnO4, (2) Polyamide Film (PF) bag with macro-perforations or (3) without a bag (control fruit). Fruit were held for 1, 2 or 3 weeks at 10°C before being unpacked, ripened and assessed for quality. On day 6 after outturn, fruit with the highest overall quality were those held in LDPE bags. LDPE fruit generally coloured up faster at outturn than PF or control fruit, had less overall moisture loss and scored high in flavour. Headspace carbon dioxide and oxygen concentrations within the LDPE bags were also near recommended levels for maintaining optimum storage-life quality. The LDPE bag provided the most suitable conditions for long term storage of fresh papaya fruit and is therefore the recommended MAP type for use with sea freight export out of Fiji.