Diagnostic Development for Canine Distemper Virus Antibodies in Panthera Species

Canine distemper virus (CDV) is a highly contagious and potentially fatal negative single stranded ribonucleic acid (-ssRNA) virus. CDV causes gastrointestinal and respiratory distress, leukopenia, hyperkeratosis and neurological symptoms and then death. Canis lupus familiaris was the only host species susceptible to CDV, but has spilled over to at least twenty mammalian species, including Panthera genus. Several species of the Panthera genus are classified as endangered by the International Union for Conservation of Nature (IUCN). CDV outbreaks has threatened stable and endangered populations with risk of extinction to several Panthera genus species. This project sets out to develop a diagnostic tool to detect CDV antibodies in serum samples using antigens produced by sections of the CDV hemagglutinin protein sequence. The work reported here details the design of the antigen sequence and cloning into an appropriate expression vector, expression of the protein and subsequent approaches to purify and recover this in a soluble form; protein characterisation and investigations into the effectiveness of the antigen when exposed to protein A/G and dipstick immunoassay studies. Traditional and Golden Gate assembly molecular cloning approaches, constructed the CDV hemagglutinin gene sequence with a C-terminus His-tag into a pET expression vector. The generated plasmid was transformed and the target antigen proteins expressed in E. coli. Several purification strategies were investigated to recover the protein from E. coli. The protein antigens investigated were largely insoluble in the absence of chaotropic agents (e.g. urea) and formed inclusion bodies; this property was used for initial crude purification of the antigen through recovery of the inclusion bodies and subsequent solubilisation in urea and provided the highest concentration of antigens. Mass spectrometry of the purified protein confirmed the correct expressed CDV hemagglutinin sequence. Immunoblotting and densitometry showed detectable signal between the antigen and Protein A/G HRP conjugate. However, ELISA and dipstick immunoassay presented challenges in coating the antigen to the surface of the well/membrane, that require future work. In conclusion, a dot blot approach showed a good correlation between antigen presence and antibody and could be utilised in the field as an alternative approach. CDV antibodies in canid serum samples is detectable with Protein A/G HRP conjugate within a laboratory and shows potential for detecting CDV antibodies in other mammalian serum samples. Development of a lateral flow assay would provide rapid detection of CDV antibodies in habitats of susceptible species.