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A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-forpurpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs). | 2495 BATH eT Al.

Feline morbillivirus (FeMV) is an emerging RNA virus in the Paramyxoviridae family that was recently discovered in domestic cats ( Felis catus). To date, 2 genotypes (FeMV-1 and FeMV-2) have been detected in cats from various countries, and FeMV-1 is recognized as a pathogen associated with nephritis. However, information regarding the pathological roles and potential transmission to other felids is limited. In this article, we describe the identification of FeMV in 2 black leopards ( Panthera pardus) in Thailand that showed severe azotemia and tubulointerstitial nephritis. Molecular analysis of the partial coding sequence of the L gene revealed that these leopard FeMV strains were genetically close to the FeMV-1 isolate from domestic Thai cats. Immunohistochemistry and immunofluorescence analyses using polyclonal IgG antibodies against the FeMV matrix (M) protein showed FeMV-M antigen in renal tubular epithelial cells. These analyses also showed infiltrating lymphocytes in the renal parenchymal lesions and in the cytoplasm of lymphoid cells residing in the spleen, suggesting viral tropism and a possible pathological role. These findings are the first evidence that indicates that the black leopard could be a possible host for FeMV infection. As for other cats, the role of FeMV as a potential cause of renal disease remains to be established. The pathogenesis of FeMV infection in black leopards, or in other wild felids, through a viral transmission mechanism warrants further investigation.

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