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A newly formatted enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bluetongue virus (BTV) was developed and validated for bovine and ovine sera and plasma. Validation of the new sandwich ELISA (sELISA) was achieved with 949 negative bovine and ovine sera from BTV endemic and non-endemic areas of Australia and 752 BTV positive (field and experimental) sera verified by VNT and/or PCR. The test diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 99.70% and 99.20%, respectively, for bovine sera, and 97.80% and 99.50%, respectively, for ovine sera. Comparable diagnostic performances were noted for the sELISA compared to four competition ELISAs. While the sensitivity of the sELISA remained unaffected by BTV-15 positive sera, the cELISAs were not as sensitive. BTV-15 is endemic to Australia, and early warning depends on sensitive diagnoses of all serotypes: endemic or incurring. The sELISA failed to discriminate against epizootic hemorrhagic disease virus (EHDV) antibodies, the most serologically related orbivirus to BTV. The ACDP cELISA and the IDEXX kit showed cross-reactivity with some EHDV serotypes, with the least cross-reactive being the VMRD and the IDVet kits. Cross-reactivities, however, were also detected in sera raised experimentally from 10 isolates of the 21 known non-BTV orbiviruses. In this case, the sELISA was the least affected, followed equally by the VMRD and IDVet kits, and the IDEXX kit and the ACDP cELISA were the least discriminatory. In addition to exclusivity assessment of the ELISAs, an inclusivity assessment was made for all ELISAs using well characterized reference sera positive for antibodies to all serotypes BTV-1 to BTV-24.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Background The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. Results Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested. Conclusions Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Background Effective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production. Results Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa) and Mycobacterium bovis (PPDb). Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition. Conclusions This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32% .

SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed significantly reduced mobility of the acylated proteins in comparison with their native forms, suggesting increased net negative charges in the acylated proteins. In contrast, the amidated proteins migrated faster than the native proteins.