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Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Purpose The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). Methods NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1–144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. Results Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. Conclusion This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.

A 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione 2 transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant 2 protein purified and its kinetic properties determined. The predicted protein consisted 3 of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. 3 Multiple alignments of the protein sequence of TcGST with homologues from other 3 helminths showed that the highest identity of 53-68% with haem-binding nematode 3 proteins designated as members of the nu class of GSTs. Substrate binding sites and 3 conserved regions were identified and were generally conserved. The predicted 3-3 dimensional structures of TcGST and HcGST revealed highly open binding cavities 3 typical of this class of GST, considered to allow greater accessibility to diverse 3 ligands compared with other classes of GST. At 25 o C, the optimum pH for TcGST 3 activity was pH 7, the V max was 1535 ± 33 nmoles.min -1 .mg -1 protein and the apparent 3 K m for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean 4 ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not 4 nematode-naïve, sheep, recognised recombinant TcGST in enzyme-linked 4 immunosorbent assays. The recognition of the recombinant protein by antibodies 4 generated by exposure of sheep to the native enzyme indicates similar antigenicity of 4 the two proteins. These findings could aid in the design of novel drugs and vaccine 4 antigens for economically important parasites of livestock.

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